Houda Zeghilet
email: hzeghilet@mail.cerist.dz

Moufida Maimour
email: moufida.maimour@cran.uhp-nancy.fr

Nadjib Badache
email: badache@mail.cerist.dz

Francis Lepage
email: francis.lepage@cran.uhp-nancy.fr

Claude Bernard

On the Use of Passive Clustering in Wireless Video Sensor Networks 1 On the Use of Passive Clustering in Wireless Video Sensor Networks

Keywords: Wireless Video Sensor Networks, routing, passive clustering, energy efficiency, load balancing, network lifetime

Wireless Video Sensor Networks are foreseen to be of a paramount importance in realizing a wide spectrum of applications, mainly surveillance, target tracking and environment monitoring. In this paper, we propose a combination of an improved clustering algorithm and directed diffusion, a well-known data-centric routing paradigm in sensor networks. On the one hand, clustering allows to save bandwidth required by rich and intensive data of video applications. On the other hand, the network lifetime is prolonged by implementing an energy-aware load balancing feature through modifying passive clustering rules for building/maintaining its topology. We performed extensive computer simulations and show in this paper that our solution outperforms original directed diffusion as well as when it is combined with passive clustering with energy considerations but without load-balancing.

Sink Internet

Figure 1 Typical Wireless Video Sensor Network

Introduction

Recent technological advances have led to the emergence of small low-power devices that integrate sensors with on-board processing and wireless communication capabilities. Pervasive networks of such sensors open new vistas for a wide spectrum of applications [START_REF] Akyildiz | A survey on sensor networks[END_REF]. Nowadays, these devices can be equipped with low-cost and low-power audio and visual modules allowing for fostering the development of Wireless Multimedia Sensor Networks (WMSN) [START_REF] Akyildiz | A survey on wireless multimedia sensor networks[END_REF]. These latter provide significant benefit to many sensor networking applications such as surveillance, target tracking, environmental monitoring, and traffic management systems. Wireless Video-based Sensor Networks (WVSN) are particular instances of WMSN where the scalar WSN is strengthened by introducing the ability of retrieving richer information content through image/video sensors [START_REF] Rahimi | Cyclops: in situ image sensing and interpretation in wireless sensor networks[END_REF][START_REF] Wu Chi Feng | Panoptes: A scalable architecture for video sensor networking applications[END_REF]. A WVSN can operate in an ad-hoc manner and hence does not require a network infrastructure adding a much higher level of flexibility and allowing a wider range of applications. Figure 1 depicts a typical WVSN architecture where video sensors are deployed at strategic positions with other non visual sensors. A central controller or a base station commonly referred to as the sink is responsible for requesting/analysing sensed data. All nodes collaborate to ensure a given application requirements. For instance, low-power scalar sensors only take part in relaying in addition to sensing environmental data. Sensors with higher capabilities could do more such as taking part in a distributed compression task in order to not overwhelm video sensors by all the tasks (capture, compression and transmission).

WVSN generate unique challenging problems and should be designed to satisfy limited resources while providing a good video quality. WVSN applications require large amount of data to be transmitted with high reporting rates which consume an order of magnitude of resources, such as storage, computation, bandwidth and especially energy. Although these issues are abundantly studied in WSN, research is still in the earlier stage in WVSN and few works are accomplished.

Protocols in sensor networks rely heavily on flooding to discover routes and deliver data. Directed Diffusion (DD) [START_REF] Intanagonwiwat | Directed diffusion for wireless sensor networking[END_REF]] is a well-know data-centric routing protocol that deals with lack of global addressing using flooding. This mechanism, although simple and effective, can be quite inefficient because of large amount of redundant messages. These latter consume scarce resources such as bandwidth and particularly energy, an important design issue in WSN. Hence energy efficient routing protocols are highly required motivating the proposition of many new routing protocols [START_REF] Akkaya | A survey of routing protocols in wireless sensor networks[END_REF].

Hierarchical routing protocols based on clustering techniques have been proposed to achieve scalability and reduce the need for global coordination. Clustering is the method by which sensor nodes in a network organise themselves into groups according to specific requirements or metrics. Each group or cluster has a leader referred to as clusterhead (CH) with possible one or more nodes belonging to at least two clusters called gateways (GW) in addition to other ordinary member nodes. As opposed to a flat organisation, clustering techniques allow more scalability, less consumed energy and thus longer lifetime for the whole network. In fact, most of the sensing, data processing and communication activities can be restricted within clusters. Moreover, clustering allows data aggregation which reduces congestion and energy consumption and can provide load balancing if appropriately configured. Furthermore, they can be naturally combined with data-centric routing to make use of data aggregation techniques.

Passive clustering (PC) [START_REF] Taek | Efficient flooding with passive clustering (pc) in ad hoc networks[END_REF] is a way to perform on-demand clustering to eliminate control messages overhead. It does not use any explicit control messages to maintain clusters. Instead, it relies on control information piggybacked on outgoing data packets. In WSN, PC was combined with DD in [Handziski et al., 2004, Rangaswamy and[START_REF] Rangaswamy | Enhancement of passive cluster based routing protocol for mobile adhoc networks[END_REF] mainly to achieve energy efficiency. To determine a routing path, DD makes use of flooding in its different phases namely: interest propagation and exploratory data sending. Therefore, the main idea of the combination is to save energy in the flooding phases by allowing only clusterheads and gateways to take part in these phases. Ordinary nodes are only allowed to send data messages in the data sending phase. In [START_REF] Md | Energy conserving passive clustering for efficient routing in wireless sensor network[END_REF], the selection of clusterheads and gateways is based on residual energy. They also proposed to apply a periodic sleep and awake among cluster members with necessary synchronisation among nodes.

All the previously cited works [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF][START_REF] Rangaswamy | Enhancement of passive cluster based routing protocol for mobile adhoc networks[END_REF][START_REF] Md | Energy conserving passive clustering for efficient routing in wireless sensor network[END_REF] concentrate traffic on a set of nodes performing flooding. We argue that this concentration can lead to a variance in energy consumption among

Copyright c 2009 Inderscience Enterprises Ltd.

sensor nodes and is able to cause rapid partition of the network. To overcome this problem, we propose in this paper to alternate flooding nodes role (clusterheads and gateways) among nodes depending on their level of battery. A node with more energy is more likely to become and keep the role of a clusterhead or gateway. In this way, a load-balancing among nodes for data dissemination is achieved with higher lifetime for the whole network. This is even more interesting in the context of WVSN where a big amount of data is to be handled.

Our changes applied on DD showed that our mechanism outperforms DD and its PC combination proposed in [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF] in terms of network lifetime and delivery ratio. We mainly studied the performances of video transmission over WSN using our approach and show how the video quality can be enhanced using a clustering algorithm along with DD. In the literature, there are some few research work trying to enhance DD so to be more suitable to video transmission but, to the best of our knowledge, none made use of passive clustering.

This paper is organised as follows. Related work is presented in Section 2. Section 3 gives details of our proposal, the Energy Level-based Passive Clustering (ELPC). Simulation results are presented in Section 4 before concluding.

Related Work

Despite the great potential held by WVSN applications, only few ones can be found in the literature. Some visual systems for WVSN were proposed in [START_REF] Rahimi | Cyclops: in situ image sensing and interpretation in wireless sensor networks[END_REF][START_REF] Wu Chi Feng | Panoptes: A scalable architecture for video sensor networking applications[END_REF][START_REF] Chu | Distributed attention for large video sensor networks[END_REF][START_REF] Kulkarni | Senseye: A multi-tier camera sensor network[END_REF][START_REF] Gerla | Multimedia streaming in largescale sensor networks with mobile swarms[END_REF]. [START_REF] Kulkarni | Senseye: A multi-tier camera sensor network[END_REF] described the design and implementation of SensEye, a multi-tier network of heterogeneous wireless nodes and cameras. The surveillance application consists of three tasks: object detection, recognition and tracking. Multimedia transport over WSN is addressed in [START_REF] Gerla | Multimedia streaming in largescale sensor networks with mobile swarms[END_REF] where a hierarchical network infrastructure is proposed to handle high bandwidth and low delay requirements of multimedia data by means of deploying a limited number of high capacity mobile nodes called swarms. [START_REF] Aghdasi | An energy-efficient and high-quality video transmission architecture in wireless video-based sensor networks[END_REF] proposed EQV-Architecture (Energy-efficient and high-Quality Video transmission Architecture) for video transmission in WSN. EQV consists of a prioritised video compression protocol in the application layer, a real time transport layer and a single-path routing protocol. Video packets are sent according to their priority and FEC are used in the link layer to achieve reliability. Another work [START_REF] Klein | Performance study of a video application over multi-hop wireless networks with statistic-based routing[END_REF] addressed frequent topology changes through the use of Statistic-Based Routing (SBR).

In this work, we are interested in applying Passive Clustering (PC) techniques on Directed Diffusion (DD) while considering energy and whole network lifetime. In what follows, both DD and PC are summarised.

Directed Diffusion

Directed Diffusion (DD) [START_REF] Intanagonwiwat | Directed diffusion for wireless sensor networking[END_REF] is the most popular data-centric routing protocol in the literature. Data-centric routing protocols are proposed to deal with the lack of a globally assigned identifiers scheme in WSN. The main idea is that data is routed based on its content rather than using routes based on unique identifiers of nodes in the sensor network. Directed Diffusion aims at diffusing data through sensor nodes by using a naming scheme for the data. It is querybased and suggests the use of attribute-value pairs for the data. In order to create a query, the sink sends an interest defined using a list of attribute-value pairs such as name of objects, interval, duration, geographical area, etc.

The interest is broadcast by a sink through its neighbours. Each node receiving the interest caches it for later use. As soon as a sensor node detects an event that matches one of the interests in its cache, it calculates a gradient for each neighbour node that delivers the matching interest. Thus, the gradients are setup from sensors to the sink. A gradient is a reply link to a neighbour from which the interest was received. Hence, by utilising interest and gradients, paths are established between sink and sources. The sink reinforces one or more paths by sending the same interest on the selected paths with a higher event rate. In addition to route discovery mechanisms, in-network processing may be employed to aggregate data to increase efficiency.

The on-demand nature of DD in constructing paths enables robustness and energy saving. Data caching and aggregation also make big benefit in terms of energy consumption and delay reduction. However, this latter feature is difficult to be applied to multimedia traffic. On the one hand, the matching process for data and queries might require some extra overhead at the sensors. On the other hand, only simple aggregation functions like averaging and thresholding are possible and are not applicable to multimedia flows that require more complex aggregation functions. The main drawback of DD, making it unsuitable to WMSN, is that it relies heavily on flooding to build and maintain paths. This is very expensive regarding required resources and can be more marked in dense networks. Finally, DD can not support time-sensitive traffic nor perform energybalancing to increase network lifetime. This is due to the fact that it makes use of the same small set of paths in the routing phase which can lead to the exhaustion of nodes on these paths and cause network partition.

It is worth mentioning that there were some few work that addressed extending DD to fit multimedia applications in WSN. For instance, authors of [START_REF] Li | Delay-constrained high throughput protocol for multi-path transmission over wireless multimedia sensor networks[END_REF] proposed a multipath extension of DD where multiple routes are reinforced based on link quality and latency. The resulting algorithm is used for transmitting video traces generated by Multiple Description Coding [START_REF] Goyal | Multiple description coding: Compression meets the network[END_REF].

Hierarchical Routing and Passive Clustering

Hierarchical protocols [Heinzelman et al., 2000, Younis and[START_REF] Younis | Heed: A hybrid, energyefficient, distributed clustering approach for ad hoc sensor networks[END_REF] save more energy compared to data-centric ones, are almost completely distributed and require no global knowledge of network. However, dynamic clustering brings extra overhead which may reduce the gain in energy consumption. Once again and like data-centric protocols, in-network processing at the cluster-heads is not practical for multimedia traffic especially in the presence of increased local communication.

In [START_REF] Qin | Maximizing video sensor network lifetime through genetic clustering[END_REF] the authors proposed a novel communication protocol for studying the upper bounds on the lifetime of a video sensor network. They proposed to organise the sensors into clusters and a linear programming model is introduced for calculating a clusterhead rotation schedule. To verify the performance of the approach, the proposed scheme was compared to other clustering algorithms in sensor networks. Simulation results showed that this solution can extend the lifetime of a sensor network up to five times over that of existing approaches when the scale of the network is not very large. The proposed cluster formation algorithm is centralised and performed at the base station or at special node which is not suitable for wireless sensor networks.

Passive clustering (PC) [START_REF] Taek | Efficient flooding with passive clustering (pc) in ad hoc networks[END_REF]] is an on demand clustering algorithm. It constructs and maintains the cluster architecture based on outgoing data packets piggybacking cluster related information. Passive clustering eliminates setup latency and major control overhead of traditional clustering protocols by introducing two innovative mechanisms for the cluster formation: "first Declaration wins" rule and "gateway selection heuristic". With the "first Declaration wins" rule, a node that first claims to be a clusterhead rules the rest of nodes in its clustered area. The "gateway selection heuristic" provides a procedure to elect the minimal number of gateways.

The algorithm defines several states in which a node can be. At cold start, all nodes are in the initial state. Nodes can keep internal states such as clusterheadready or gateway-ready to express their readiness to be respectively a clusterhead or gateway. A candidate node finalises its role of a clusterhead (CH), a gateway (Full-GW or Dist-GW) or an ordinary node. Additional fields suggested by PC in the message header of each packet are :

• id : the identity of the originator of this message,

• state : this packet sender status in the network,

• CH1 and CH2 : one (or both of them) is (are) used by a gateway to announce its clusterhead(s) id(s),

The reactive nature of PC motivated its combination with on demand routing protocols. Originally, PC was applied to reactive unicast (AODV [START_REF] Das | Ad hoc on demand distance vector (aodv) routing[END_REF], DSR [Johnson et al., 2001]) and multicast (ODMRP [START_REF] Lee | On-demand multicast routing protocol (odmrp) for ad hoc networks[END_REF]) routing protocols. The major overhead in these routing protocols is caused by the flooding of route queries. It was suggested to allow only non-ordinary nodes to rebroadcast query messages.

The PC algorithm presents some shortcomings that have been targeted by several works. In [START_REF] Rangaswamy | Enhancement of passive cluster based routing protocol for mobile adhoc networks[END_REF], the authors proposed to add alive packets to keep the cluster stability as it depends highly on the data packet traffic. Also, a sequence numbering to synchronise packets arriving from a source node is proposed. In fact, if packets containing different states arrive out-of-order at the destination (i.e., the sending node changed its state between transmission of multiple packets) then the destination node will be misled about the true state of the source node. In addition, unnecessary rebroadcasts are eliminated when the final destination of the message is a cluster member.

In WSN, the PC algorithm was proposed in combination with directed diffusion [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF] to mainly achieve energy efficiency. The main idea of the combination is to save energy in the flooding phases by allowing only clusterheads and gateways to participate in them. Ordinary nodes are only allowed to send data messages in the data sending phase. Under different network sizes and loads, the combination when compared to DD, showed better performances in terms of delivery ratio and average dissipated energy.

Motivated by the results shown in [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF] when applying the original PC along with directed diffusion paradigm other work have been proposed in order to achieve better performance of the combination.

In [START_REF] Md | Energy conserving passive clustering for efficient routing in wireless sensor network[END_REF], the selection of clusterheads and gateways are done using a heuristic of residual energy and distance. By using residual energy the flooding nodes are chosen in an energy efficient manner. Distances are used to reduce the overlapping region and so the number of gateways. The solution proposed to apply a periodic sleep and awake among cluster members. This technique is similar to the one proposed in LEACH [START_REF] Heinzelman | Energy-efficient communication protocol for wireless microsensor networks[END_REF] and requires a synchronisation process between nodes.

Energy Level-based Passive Clustering (ELPC)

The main idea in combining PC to DD is to reduce energy consumption by minimising flooding. As this process is known to be very costly, the energy expenditure of the flooding nodes will be much higher than those of ordinary nodes. This will cause a variance in the power amounts of the nodes in the network and by that a fast partitioning of the network. This is the case of all previously cited works where there is no loadbalancing feature when combining PC to DD. In fact, topology construction in PC is done according to the lowest ID. The drawback of doing so is its bias towards In this work we propose ELPC (Energy Level Passive Clustering) to achieve energy efficiency in terms of network lifetime, not only in terms of energy consumption. This is done through alternating flooding nodes role (clusterheads and gateways) among nodes depending on their energy. The aim of doing so is to have the same amount of energy at all the nodes at a given time which increases substantially the whole network lifetime.

In ELPC, each node's battery is split into levels. One can make a correspondence between different energy levels of a node and virtual sub-batteries it consumed sequentially. The energy level (l) of a node can be computed using :

l = L E r E i (1)
where E r is the remaining energy, E i is the initial one and L is the suggested number of levels. For instance, if the number of levels is equal to 5, a node with only the half of its battery will have an energy level of 3.

We introduce the notion of candidature to be a clusterhead or a gateway by defining the network energy level (nel) parameter. A node is not allowed to declare itself as a clusterhead (or a gateway) if its energy level is lower than this parameter. A clusterhead (or a gateway) can keep its role as long as its energy level is higher than the nel. Otherwise, it gives up its role and passes to the initial or ordinary state according to whether it knows or not a clusterhead in its vicinity.

Finding a meaningful value for the network energy level is non-trivial. It depends on the energy level of the network nodes and can be viewed as the minimum level of energy necessary for a node to be a clusterhead or a gateway. We suggest to take an initial value that We use the same states as suggested in [Kwon and [START_REF] Taek | Efficient flooding with passive clustering (pc) in ad hoc networks[END_REF] where a node is initially at the initial state. Nodes form and maintain the clustering topology by changing their internal and external states based on incoming and outgoing messages. When sending the next message, a node announces its external state which becomes visible in the network. Algorithm 1 summaries how PC is modified to allow load-balancing feature depending on nodes energy levels.

In addition to the PC related fields, we add the following ones to the packet headers (Figure 3) :

• l, node's energy level

• nel, the network energy level

• give-up, as in [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF] is set when the node is a CH that gives-up its role. It is used to replace the give-up message proposed in [START_REF] Taek | Efficient flooding with passive clustering (pc) in ad hoc networks[END_REF]. In ELPC, this field is set when the energy level of a CH drops bellow the nel.

Figure 4 illustrates the same example of Figure 2 with ELPC applied. The number of levels is chosen to be five for all the three nodes and the nel is initially set to 3 corresponding to the half of battery charge. We can see that the clusterhead role is alternated between the three nodes depending on their energy levels. When two nodes have the same energy level, then the nodes' identities are used to solve conflict in declaring roles. At step 3, we can Algorithm 1 ELPC

• CH list is the list of known clusterheads to this node,

• GW list is the list of known gateways to this node,

• ORD list is the list of known ordinary nodes to this node,

• IN IT list is the list of known nodes to this node that are in initial state,

• give up if set, it indicates that this node wants to give-up its role, note that node 1 decreases its nel to 2 (since l = nel) and propagates this new value to its neighbours so all nodes can have same estimation of the network energy level. It is straightforward that using ELPC compared to PCDD (Figure 2), we enhance the network lifetime by allowing fair energy distribution among nodes.

Initialisation
Figure 5 shows the establishment of routing structures of directed diffusion when this latter is used in combination with ELPC. Initially, all nodes in the network are in the initial state. Nodes will use the first interest messages to establish the new topology as described in the algorithms. A possible topology is illustrated in Figure 5(a-b). After establishing the gradient (Figure 5(c)) and path reinforcement (Figure 5(d)), the source begins sending the sensed data. When the energy level falls under the network energy level at node A, it gives-up its role of clusterhead (Figure 5(d)). Thus, a new topology is established (Figure 5(e)). This is done using next circulating messages in the network (data messages, interests, exploratory data). The resulting passive clustering can be applied to any routing protocol in sensor networks as they mostly rely on flooding and particularly with DD. This not only reduces energy consumption as in [START_REF] Handziski | Improving the energy efficiency of directed diffusion using passive clustering[END_REF], it also increases the whole network survivability as it will be shown in section 4.

Before presenting the simulation results, we show using a simple formal method how ELPC allows longer lifetime than PCDD. A more accurate and developed formal method is beyond the scope of this paper. Consider a network region with one cluster and say k, k ≥ 1 potential candidates to be a clusterhead. Let E L be the amount of energy per level which we consider to be the same for all the region sensors. Let E i be the available energy at a sensor i in the beginning of the session where E 1 ≤ E 2 ≤ ... ≤ E k-1 ≤ E k . We call a round the time interval in which a given candidate is a clusterhead. Let P be the power dissipation of a sensor when it is a clusterhead.

In PCDD, the number of rounds is k since each candidate becomes a clusterhead once. Each round lasts E i /P when sensor i is the clusterhead in this round. Given that a region lifetime is defined as the time until the first candidate dies, the PCDD lifetime corresponds to the duration of the first round. Since, it is the candidate with largest amount of energy (k) who is elected for the first round, then this region lifetime in PCDD can be given by :

Λ P CDD = E k P (2)
In ELPC, the number of rounds is k i=1 E i /E L and each candidate (i) becomes a clusterhead E i /E L times. This is because each round consists in exhausting a level (E L ) of energy. It comes that each round lasts E L /P . When the first clusterhead is died (the kth one with the largest amount of energy at the beginning of the session), it remains one level of energy for the (k -1) other candidates and this region lifetime can be computed as follows :

Λ ELP C = E L P ( k-1 i=1 (E i /E L -1) + E k /E L )
Equivalently :

Λ ELP C = 1 P ( k i=1 E i -(k -1)E L ) (3)
Note that E i = E L when L = 1 and that :

∀i = 1, k -1 : E i ≥ E L
Then we can write the following :

k-1 i=1 E i ≥ (k -1)E l k i=1 E i ≥ E k + (k -1)E l
Dividing by P , it follows that Λ ELP C ≥ Λ P CDD and that Λ ELP C > Λ P CDD when L > 1 and k > 1. This means that if we have only one level of energy (L = 1) or just one potential candidate to be a clusterhead (k = 1), ELPC behaves exactly like PCDD. To get better performances in ELPC, we need naturally to have more than one level of energy and more than one potential candidate so the network connectivity is ensured for longer time.

Simulation Results

We implemented our energy-level passive clustering (ELPC) using NS-2 [ns2] and compared it to the original directed diffusion (DD) and PCDD (DD combined to passive clustering with energy considerations but without load balancing feature as done in [START_REF] Md | Energy conserving passive clustering for efficient routing in wireless sensor network[END_REF]). We used the Two Phase Pull diffusion algorithm with its two flooding phases. The interest flooding is initiated by the sink and the data messages flooding is performed by the the sources upon event detection in the network. Additional fields in the message header are added as attributes to represent the node's energy level and the network energy level.

For all the experiments conducted in this paper, the sensors field is of 160 × 160 m2 with a varied number of nodes ranging from 100 to 500 nodes. We used the IEEE 802.11 and the two-ray propagation model. The radio propagation of each sensor node reaches up to 40 meters. We took the same amount of energy per level (5 J) at all nodes so the number of energy levels depends on the initial amount of energy at nodes. Results are averaged over 20 randomly generated topologies. We performed both sufficient and insufficient energy scenarios. In the first set, nodes have sufficient amount of energy to terminate the simulation (the source is able to send all the video clip considered). This allows us to mainly assess the average dissipated energy per correctly received information. In the second set of simulations the amount of energy at nodes is chosen so it is smaller than the minimum required energy in the three protocols. This allows us to assess the network lifetime in seconds. This latter is obtained using the time at which the sink receives the last data packet from the video source.

To evaluate the quality of a video transmitted over our WVSN, One video sensor is assumed to capture and transmit a video sequence. We selected one of the standard video sequences used by a variety of video encoding and transmission studies called "Hall Monitor". It lasts 10 seconds and consists of 300 frames in CIF resolution (352 × 288). The video sequence is encoded in MPEG4 using ffmpeg [ffm] with a target bit rate of 128Kbps and a Group of Pictures (GOP) of 30. Only I (intra) and P (predicted) frames were generated in video traces using the open source EvalVid set of tools [START_REF] Klaue | Evalvid -a framework for video transmission and quality evaluation[END_REF]. The reference (or the sent video) PSNR (Peak Signal to Noise Ratio) obtained is 29.70 dB.

We considered the evaluation, in terms of PSNR, of the received video quality to show the benefit of passive clustering to video applications in WSN. The PSNR between the sent (s) and the received (r), possibly distorted video sequence is computed using :

P SN R(s, r) = 20log V peak M SE(s, r) (4)
M SE is the mean square error which is the average of the square of the errors (pixel differences) of the two images and V peak is the maximum possible pixel value.

We conducted experiments in order to empirically find the optimal data packet size for a good trade- off between energy efficiency and video quality. We considered a 200-node network and vary the data packet size from 32 to 2048 bytes. We obtained similar results for both insufficient and sufficient energy scenarios. We chose to present here only curves related to insufficient energy experiments. Figure 6 plots the loss ratio for the three simulated protocols (DD, PCDD and ELPC) as a function of data packets size. It is seen that the performances show a drop for packets size around 128 bytes. This behaviour can be attributed to the higher packet drops caused mainly by collisions for smaller packet sizes as bigger number of packets are to be sent. Even if the number of lost packets is smaller for large packet sizes, these losses affect considerably the loss ratio as the number of packets to be sent is smaller.

Our experiments show that the empirical optimal data packet size is 128 bytes which corresponds to the smallest loss ratio. This value is confirmed in video quality measurements and network lifetime shown in Figures 7 and8 respectively. We can see that the maximum PSNR and lifetime for all the three protocols are obtained for data packets of 128 bytes. These experiments confirm in someway the results presented in [START_REF] Sankarasubramaniam | Energy efficiency based packet size optimization in wireless sensor networks[END_REF] where it is shown that for a given bit error probability, energy efficiency decreases when packet size exceeds a threshold which is nearly 100 Bytes. At this stage, it is worth mentioning that ELPC presents the best performances regardless the data packet size used.

In all what follows, we will use data packet size of 128 bytes. In subsequent simulations, the video clip is sent twice. This is done in order to get more insight into both the transient and steady phases of passive clustering in PCDD and ELPC. Each simulation runs until network partition (no way to reach the sink from the video source) or all the data packets composing the two clips are received by the sink. We start by giving the overall performances and then those related to the first and second clip periods.

Overall Performances

We are interested here in overall performances related to the entire simulation time from sending the first packet of the first clip until the last packet of the second clip. Figure 9 shows the energy gain obtained in PCDD and ELPC with respect to DD with sufficient energy. Passive clustering allows energy saving in both ELPC and PCDD where they at most consume 45% of what DD consumes for 100-node network. When increasing the network size, the gain is much improved mainly using ELPC (more than 85% of energy is saved compared to DD for 500 nodes). This is because in ELPC more data packets are delivered than in PCDD as will be shown later on.

The improvement is mainly due to the fact that clustering reduces the number of floods in the network and thus nodes will consume smaller amount of energy. This helps in extending network lifetime. In ELPC, an alternation of the roles is achieved. Clusterheads or gateways give up their roles when other nodes with higher energy levels are in conflict with them or when their energy level is lower than the network energy level (nel). This encourages other nodes to declare themselves for these roles and, by that, leads to an increasing in the nodes lifetime. As a consequence, the lifetime of the whole network is extended. The nodes of the network are then able to forward more data, which, permits to the sink to receive a higher number of events.

Prolonging the network lifetime is our primary goal when proposing ELPC. We conduct experiments with nodes assigned insufficient energy to capture network lifetime. We use as a lifetime metric, the duration of the video received by the sink. Figure 10 plots as the network size increases, the network lifetime for the three protocols with insufficient energy. The network lifetime slightly decreases with the number of nodes increasing since a larger number of nodes results in a higher number of floods and hence bigger amount of energy is consumed in the network. Our solution (ELPC), however, achieves better performances compared to the two others. In ELPC, almost the two clips are received and the network lifetime is estimated to be nearly 20 seconds, the two clips duration. However PCDD and DD best lifetime does not exceed 13 and 9 seconds respectively.

Regarding video quality, ELPC outperforms both DD and PCDD mainly for insufficient energy scenarios as shown in Figure 11. Video quality decreases with

First Clip Period

Here, we are interested in studying the transient period of the three protocols mainly PCDD and ELPC since clusters formation needs a given period of time to completely converge (the clustered topology is entirely built). It is worth saying that first clip related results (mainly for PCDD and ELPC) are nearly the same for both sufficient and insufficient energy scenarios since the amount of available energies are chosen (even in insufficient energy case) so at least a minimum number of the first clip frames are received by the sink.

Figure 13 shows the video mean PSNR as the network size increases. We can observe that ELPC and PCDD present relatively same performances. This is was to be expected since ELPC is likely to perform better when there is lack of energy in the network as will be shown in section 4.3. Figure 14, showing loss ratio of the three protocols as a function of the network size, confirms these results. We can see that ELPC and PCDD achieve roughly similar results which are clearly better than the ones given by DD. ELPC achieves better delivery ratio than PCDD in most cases however as already seen in Figure 13, it presents lower PSNR. This can be explained by the fact that in the performed set of experiments, there were more lost I-frames packets in ELPC. There is no way to distinguish I and P frames in all the three simulated protocols.

Figures 15 and16 show the evolution of PSNR in per frame basis for one scenario with respectively 100 and 500 nodes for the three protocols as well as the reference PSNR (REF). This latter corresponds to the measured quality of the sent video and allows to consider only network effects on the video quality. It is clear that both PCDD and ELPC outperforms DD thanks to flooding reduction through clustering.

Clustering allows to achieve similar PSNR values as the reference PSNR except for the first 30 frames where ELPC and PCDD performances are as bad as those of DD. These 30 frames correspond exactly to the first transmitted GOP. Their bad quality can be explained by the presence of a transient period where The mean PSNR obtained with ELPC is of about 26.31 dB for 100-node network while it is of only 16.03 dB for DD. Note that the mean PSNR of the transmitted video is 29.7 dB. DD is unable to reach a PSNR greater than 18 dB which is very bad as can be observed in Figure 17. For a 500-node network, similar results are obtained when comparing the three protocols however with lower PSNR values due to scalability issues mainly for DD.

Figure 17 shows two sample images as sent (a) and received by the sink using DD (b), PC (c) and ELPC (d). Video quality enhancement of ELPC compared to DD is noteworthy. For instance, the best achieved quality in DD corresponds to the image in the left (14.1 dB) for which, we can note the bad quality especially compared to the one of ELPC (with a PSNR of 27.56 dB). This can be explained by losses caused by messages dropped due to congestion in the network as large number of flooding are performed according to DD conception.

Second Clip Period

In this section, we are mainly interested in comparing ELPC to PCDD to see how the former compared to the latter is able to enhance video quality mainly for insufficient energy scenarios. Figure 18 plots the mean PSNR obtained at the sink as function of the number of sensor nodes with sufficient energy. The first thing to note is that even with sufficient energy, DD performs very bad with a PSNR less than 19 dB. We chose to omit DD results since in many cases mainly for insufficient energy scenarios, only few frames are received from the second clip. This is why in subsequent plots, we only consider ELPC and PCDD.

As PCDD regardless of initial amount of energy available at the sensor nodes. The difference is mainly observed in insufficient energy cases. Figure 19 shows that PCDD achieves at most a mean PSNR of 20.25 for a 100-node network while ELPC mean PSNR is 27.73 dB. Even if the network size is increased to reach 500 nodes, ELPC obtains a mean PSNR of 24.27 dB.

PSNR results of Figure 19 are confirmed by the loss ratio experienced by both protocols as depicted in Figure 20. In PCDD, the larger the network, the higher the loss rate since the traffic overheard is more important. This explains why the PSNR is worser when the network size increases. Losses are more important in PCDD because of higher number of nodes ran out of energy since no energy-aware load-balancing is performed. This leads to premature network partitioning and thus shorter lifetime as already shown by Figure 10.

Figure 21 plots the mean PSNR on a per-frame basis for a 300-node network with sufficient and insufficient energy. In both cases, ELPC outperforms PCDD especially for insufficient energy simulations where ELPC achieves a mean PSNR of 25.87 dB while PCDD a mean PSNR of only 16.57 dB suffering from higher loss rates. We can see that in ELPC, the frames Figure 22 shows frames number 30, 60 and 90 as received by the sink in insufficient energy simulations in PCDD and ELPC as well as the reference ones (as sent from the source). The three frames correspond to the last frames of the three first GOPs. This allows assessing video quality using the worst frame in a GOP just before receiving a new I-frame that could increase considerably the observed PSNR. We can see that frames quality in ELPC is better than those obtained in PCDD and is of approximately the same quality of the reference frames. For instance, the frame 30 is received with only a PSNR of 14.20 dB with very bad quality in PCDD while it is received with a PSNR of 29.21 dB in ELPC.

An other important observation is that frames 60 and 90 are nearly the same for PCDD. This means that very few data arrived at the sink from the third GOP. In the corresponding simulation scenario, the last received packet using PCDD corresponds to the 69th frame of the second clip. In the same simulation scenario, no frame from the second clip is received using DD.

Conclusion

In this paper, we proposed ELPC, a combination of a well known routing paradigm (DD) in sensor networks with an energy efficient cluster formation algorithm. This combination gives better performances in terms of network survivability and data delivery ratio when small amounts of energy are available. We were mainly interested in evaluating video transport in a WVSN using passive clustering. We conducted extensive simulations and showed that an approach like ELPC with energy-aware load balancing feature is very promising. ELPC allows longer network lifetime, lower loss rate and better video quality.

In the future, we plan to carry out deeper performance studies for different types of video and network conditions. Moreover, processing capacity of video sensors have to be considered. As a result, currently, we are working on encoding methods that consume less energy. We mainly expect to study the trade-off between processing requirements (mainly in terms of energy) and video quality. Priority schemes will also be considered since I-frames packets have to get distinguished from P-frames for instance.

Figure 2

 2 Figure 2 PC without load-balancing

Figure 4

 4 Figure 3 ELPC Packet header

Figure 5

 5 Figure 5 ELPC illustrated

Figure 6

 6 Figure 6 Loss ratio

Figure 8

 8 Figure 7 Video quality (PSNR)

Figure 9

 9 Figure 9Energy ratio per correctly received byte (sufficient energy)

Figure 12

 12 Figure 10 Lifetime with insufficient energy

Figure 13

 13 Figure 13 Video PSNR -First clip

Figure 16

 16 Figure 14 Loss ratio -First clip

Figure 17 Figure 19

 1719 Figure 17 Sample of received frames (60 and 61) from the first clip : (a) Reference, (b) DD, (c) PC and (d) ELPC

Figure 21

 21 Figure 20 Loss ratio with insufficient energy

Figure 22

 22 Figure 22 Sample of received frames (30, 60 and 90) from the second clip with insufficient energy : (a) Reference, (b) PC and (c) ELPC

  phase return; {example: if the clusterhead has givenup its role,the node passes to the initial state} 8: end if 9: if msg.state == CH then add the gateway to the corresponding list and update its state {the same principle is applied. Here the conflict takes place when the states are the same and the related CH are also the same. The energy level is then used to solve it.} 26: end if 27: if (state == initial) AND (msg.state! = CH) then

	1: state ← initial 2: loop 3: wait for receiving/sending a message 4: end loop	Outgoing message processing 1: if give up == true then 2: give up ← f alse; 3: if CH list is not empty then 4: state ← Ordinary;
	Incoming message processing	5: 6:	else state ← initial;
	1: give up ← f alse	7:	end if
	2: if msg.nel < nel then	8: end if
	3:	nel ←msg.nel;	9: if state == CH Ready then
	4: end if	10:	if l > nel then
	5: if msg.give -up then	11:	state ← CH;
	6:	delete the node from lists and updates its state;	12:	else
	7:		13:	decrease nel;
			14:	if CH list is empty then
			15:	state ← CH;
			16:	end if
	10:	if state == CH then	17:	end if
	11:	if l < msg.level then	18: end if
	12:	give up ← true;	19: if state == GW Ready then
	13:	add CH to the CH list;	20:	if l > nel then
	14:	else if l == msg.level then	21:	state ← GW ;
	15:	Use nodes' identities to solve conflict (if	22:	else
		any)	23:	decrease nel;
	16:	else	24:	if GW list is empty then
	17:	add the CH to the CH list; check lists;	25:	state ← GW ;
	18:	return	26:	end if
	19:	end if	27:	end if
	20:	end if	28: end if
	21: else	29: if (state == CH) OR (state == GW ) then
	22:	add the CH to the CH list; check lists;	30:	if l < nel then
		recalculate my state;	31:	give up ← true;
	23: end if	32:	if CH list is not empty then
	24: if (msg.state == GW ) then	33:	state ← Ordinary;
	25:		34:	else
			35:	state ← Initial;
			36:	end if {the same principle is applied if the
				node is a gateway. If the first condition is
				not satisfied the node declares itself as an
				ordinary node: state ← Ordinary}
			37:	end if
			38: end if
	28:	state ← CH Ready;	39: Update msg fields; send msg;
	29: end if

  shown by Figures18 and 19, ELPC allows better video quality in terms of mean PSNR with respect to

	29.00 dB	28.950 dB
	(a)
	14.1 dB	14.1 dB
	(b)
	18.73 dB	28.9 dB
	(c)
	27.56 dB	28.90 dB
	(d)


Deepu A Gopakumar

Avinash R Pai

| Yasir

Beeran Pottathara

Daniel Pasquini

| Luís

Carlos De Morais

Abdul Khalil

Ange Nzihou

Sabu Thomas
email: sabuthomas@mgu.ac.in

Flexible papers derived from polypyrrole deposited cellulose nanofibers for enhanced electromagnetic interference shielding in gigahertz frequencies

Keywords:

An array of highly conductive, lightweight and flexible cellulose nanopapers as effective attenuators of electromagnetic radiations within 8.2-12.4 GHz (X band) were formulated via in situ polymerization of pyrrole monomers on to cellulose nanofibers (CNFs). It is quite obvious that the free hydroxyl groups on the surface of CNFs facilitate the formation of intense intermolecular hydrogen bonding with PPy which is envisaged for its excellent electromagnetic shielding performance with an average shielding effectiveness of ca. -22 dB (>99% attenuation) at 8.2 GHz for a paper having 1 mm thickness. The fabricated papers displayed a predominant absorption mechanism (ca. 89%) rather than reflection (ca. 11%) for efficiently attenuating electromagnetic radiations, which has a considerable importance in the modern telecommunication sector. Thus, the designed PPy/CNF papers would replace the conventional metal-based shields and pave way for the development of green microwave attenuators functioning via a strong absorption mechanism. The PPy/CNF nanopapers exhibited a DC conductivity of 0.21 S/cm, a prime requisite for the development of highly efficient electromagnetic shields. Undoubtedly, such nanopapers can be employed in wide range of applications such as electrodes for supercapacitors and other freestanding flexible paper-based devices.

| INTRODUCTION

Conducting filler-based polymer composites have been extensively studied for diverse applications such as electronic packing, electromagnetic interference (EMI) shielding, high-storage capacitors, and radar adsorption and so forth. [1][2][3][4][5][6] Among these, EMI shielding has gained considerable interest due to the outrageous intermixing of electromagnetic signals emitted from highly sensitive electronic instruments. The repercussion of this electromagnetic pollution not only deteriorate the life of the electronic devices but also poses serious threat to the health of human beings. 4,7 For practical EMI shielding applications particularly in household appliances, automobiles and aerospace, lightweight flexible polymer composite material has gained a considerable attention for microwave suppression due to its many advantages over the traditionally used metal sheets. [8][9][10][11][12] Intrinsically conducting polymers (ICPs) are gaining enormous interest as excellent candidates for EMI shielding due to their ease of processing, low cost, lightweight, corrosion resistance, and tuneable conductivities. [13][14][15][16] Moreover, the adsorption dominated shielding mechanism of ICPs suggests as huge potential for EMI shielding applications than metals as shielding mechanism of metals is mainly by reflection of the incident radiation. 14,17 Among the ICPs, PPy gained considerable attention due to its easy synthesis, environment friendly and high-electrical conductivity. 18 PPy has been reported as an efficient material for biomedical field, 19 pH sensors, 20 electrochromic devices, 21 antistatic materials, 22 fuel cells, 23 mechanical sensors, [START_REF] Tjahyono | Sensors Actuators[END_REF] electromagnetic shielding. 25,26 Nevertheless, the presence of the long order conjugated system in PPy increases the rigidity of the material, which leads in the poor film-forming ability. In addition, the inability of PPy in forming robust films are due to the incapability of PPy to form H bonds within the polymer network. In order to rectify this problem, considerable works have been reported on the fabrication of different PPy composites with a wide range of polymeric materials with excellent physical properties and good processability. Various polymeric materials such as polyoxyphenylene, 27 polyester, 28 polyamide, 29 cellulose, 26,30,31 rubber, [START_REF] Tjahyono | Sensors Actuators[END_REF]32 and plastic 33,34 have been extensively studied with PPy for various technological applications. Among them, cellulose nanofibers (CNFs) have drawn more interest due to its abundant, cost effectiveness and eco-friendly nature. Different forms of cellulose such as CNFs, 35 filter paper, 36 cellulose nanocrystals, 37 bacterial nanocellulose, 26 cellulose derivatives, 38,39 and cellulose aerogels 40 have been employed as a substrate for the in situ polymerization of PPy. Among the abovementioned cellulose forms, CNFbased papers have been proposed as an efficient substrate for PPy. Since CNF-based nanopapers give adequate Hbonding, the poor film-forming capability and frangibility of PPy could be rectified by fabricating flexible PPy/CNF nanopapers that can be used to shield electromagnetic radiations.

In this context, herein we produced a series of PPy/CNF conductive nanopapers via in situ polymerization of polypyrrole on CNFs to suppress the electromagnetic radiations in the X-band frequency. It is also notable that, extensive reports on the PPy/CNF conductive nanopapers as effective EMI shielding material for practical device applications are not studied and reported. So far, the only report is by Tang et al., where they fabricated a flexible conductive polypyrrole nanocomposite membranes via in situ chemical synthesis of pyrrole monomer on the surface of the bacterial cellulose and fabricated membrane had an overall shielding effectiveness (SE) of -15 dB (around 96.8% attenuation). The commercial utility of the demonstrated membrane is limited as it requires -20 dB for device applications. The key highlight of the demonstrated work is that, the fabricated PPy/CNF conductive nanopapers had accomplished an EMI (SE) of ca.

-22 dB (more than 99% of shielding), which makes them as an effective commercial EMI attenuator in the current scenario. In addition, the absorption domination shielding mechanism of the demonstrated PPy/CNF nanopapers facilitates to attenuate the electromagnetic radiations without any threats to the human beings. To the best of our knowledge, the demonstrated work is the first report on PPy/CNF conductive nanopapers with an EMI (SE) greater than -20 dB, which makes the PPy/CNF nanopaper suitable for commercial practical device applications as it surpasses the value of commercial requirement of -20 dB via adsorption dominated mechanism.

| EXPERIMENTAL

| Materials and instruments

Pyrrole (pure, d = 1.022 g/ml, Mw = 93.13 g/mol), ammonium persulfate (APS, 98% purity, Mw = 228.20 g/ mol), and hydrochloric acid (35%) were procured from Sigma-Aldrich, Brazil. The CNFs were supplied from SUZANO, Brazil. The CNFs were supplied as in water suspension which were extracted from eucalyptus bleached Kraft pulp and diameters in ranging from 20 to 30 nm. All reagents were used without any further purification.

The DC electrical conductivity of the fabricated PPy/CNF nanopapers was measured at the ambient conditions by the standard four-probe method. In order to measure the conductivity of the samples, a Keitheley employed. In order to minimize the error in the DC conductivity value, the measurements were taken in accordance with the van der Pauw method. For highfrequency dielectric measurements of the fabricated PPy/CNF paper, a Novocontrol Alpha High-Resolution Dielectric analyzer with 10 to 15 GHz resonator having the sample dimension of 3 x 3 cm was used. In order to investigate the morphology of the CNF and PPy/CNF conductive nanopapers, (CARL ZEISS, EVO MA10) scanning electron microscope (SEM) was used after sputtered with gold coating in an argon atmosphere at 20 mA for 2 min.

X-ray diffraction (XRD) patterns of the CNF, PPy, and PPy/CNF were obtained using SHIMADZU XRD-6000 at a 2θ range from 5 to 40 . Fourier transform infrared (FTIR) spectra of all samples were recorded on IR prestige-21, FTIR spectrophotometer, Shimadzu in the range of 400-4000 cm -1 . The SE of samples was measured by Agilent E8362B (10 MHz-20 GHz) two-port PNA Network analyzer attached to a Keycom waveguide to evaluate the scattering parameters at the frequency range of 8.2-12.4 GHz (X band). The SE of the samples were calculated from the scattering parameters (S11, S12, S22, and S21) using standard equations.

| Preparation of PPy/CNF suspensions (in situ polymerization)

In order to synthesize polypyrrole, in situ polymerization strategy was used in this current study. For the preparation of in situ polymerized PPy/CNF suspension with various ratios of 0.2:1, 0.3:1, 0.5:1,0.8:1, 0.9:1, and 1:1, various amounts of pyrrole monomer were dissolved in 40 g of CNF suspension (1.15 mg/ml) at ambient temperature for 1 h. After this, the prepared suspension was cooled at 1 C, subsequently APS solution (dissolved in 40 ml of 1 M HCl) was added drop wisely into the cooled suspension. After 2 h, the black suspensions of PPy/CNF were formed and suspended. Afterwards, the resultant suspensions were filtered and washed with distilled water and acetone in order to remove the oxidized components and unreacted pyrrole monomer.

| Fabrication of PPy/CNF flexible nanopapers

The resultant suspensions were vacuum filtrated via filtration assembly with a cellulose ester membrane with 0.45 μm pore size and diameter of 47 mm to fabricate PPy/CNF nano flexible paper. Finally, the fabricated nanopaper was washed with 1 M HCl in order to remove all impurities followed by drying at 70 C for 4 min in hot press.

| RESULTS AND DISCUSSION

The scheme in Figure 1 represents the formation of PPy/CNF flexible composite paper through the in situ polymerization of pyrrole on the CNFs. As demonstrated, the polypyrrole could be coated on CNF surface as shown in Figure 2 via hydrogen bonding between the nitrogen of PPy and hydroxyl group of CNFs as shown in Figure 3. Upon addition of the APS oxidant, the pyrrole monomers would be polymerized on the surface of these CNF fibers, which resulted in the formation of dark black color flexible paper. OCH in-plane bending, CH bending, C O C stretching, respectively. 41,42 In the case of PPy, strong bands arising at 3218 and 1546 cm -1 was attributed to the N H stretching vibration and C C stretching of aromatic ring of PPy. Moreover, the peaks at 1306 and 1151 cm -1 corresponds to C H in-plane ring bending and C N in-plane ring deformation respectively. 43 In the case of PPy/CNF nanopaper, a superposition of the characteristic peaks of both CNF and PPy was observed without any formation of new peaks. This infers that, there was no chemical reaction between both CNF and PPy. It is worth mentioning that the major peaks of PPy (3218, 1546, and 1157 cm -1 ) was shifted to lower wave numbers in PPy/CNF nanopapers (3206, 1544, and 1151 cm -1 ) which is ascribed to the strong intermolecular hydrogen bonding interaction between CNF and PPy. [44][45][46]

| FTIR studies

| Crystallinity studies

The XRD profiles of PPy powder, PPy/CNF and CNF nanopapers are shown in Figure 5. The neat CNF paper exhibited two diffraction peaks at 2ɵ = 16.3 and 2ɵ = 22.6 , assigned to (110) and (200) planes. The XRD pattern of cellulose type I structure was clearly reflected in the neat CNF paper. 42 The pure PPy exhibited a broad diffraction peak around 19.6 , shows its amorphous nature 47,48 The XRD spectra of PPy/CNF nanopaper shows the combination of PPy and CNF diffraction profiles. Comparing with the neat CNF paper, the intensity of the diffraction peaks was reduced in the PPy/CNF nanopaper, which confirms the uniform coating of pyrrole monomers on to the surface of CNFs during the in situ polymerization reaction.

| Surface topography and cross section of PPy/CNF flexible nanopapers

To investigate the surface topography of the fabricated PPy/CNF nanopaper SEM images was used. From the Figure 6(a), it could be seen that each CNF was merged together to form a bundle of fibers during vacuum filtration via strong intermolecular hydrogen bonding within the CNFs. Hence, it becomes practically difficult to distinguish individual CNF fibers alone from Figure 6(a). By comparing the Figure 6(a),(b), it could be clearly seen that pyrrole monomers were uniformly coated over the CNFs through in situ polymerization, and thereby resulted in the highly conductive PPy/CNF nanopapers. Even though, both neat CNF paper and PPy/CNF nanopaper exhibited similar morphology, surface roughness and diameter of CNFs was enhanced due to the uniform coating of pyrrole monomer on the surface of CNF in PPy/CNF nanopaper.

From the Figure 6(b), it was shown that the fibers were merged together which implies that, the strong hydrogen bonding interaction between nitrogen of PPy and OH group of CNFs, which was confirmed from the FTIR studies. The cross-sectional view of the neat CNF and PPy/CNF nanopaper were illustrated in Figure 7(a), (b) respectively. The cross-section of the neat CNF exhibited a layered pattern, which was attributed to the strong H-bonding among the CNFs itself. The crosssection of the both papers exhibited a similar layer pattern structure but PPy/CNF nanopaper shows some white spots illustrating the broken ends of the CNFs (see Figure 7 For the transport of mobile charge carriers, it is essential to have a continuous network of PPy on the surface of CNFs even at low concentration of PPy (0.2:1). 49,50 Table 1 illustrates the conductivity values of PPy/CNF nanopapers with different concentrations of PPy. The high aspect ratio of CNFs and the strong hydrogen bonding between the PPy and CNF had resulted in the highly conducting nature of PPy/CNF nanopapers. It is noteworthy that, the fabricated PPy/CNF nanopapers with ratio of 1:1 (PPy/CNF) showed a highest value of 0.21 S/cm. Figure 8

| Dielectric properties of PPy/CNF nanopapers

If a material exhibits decent dielectric properties with high dielectric permittivity and dielectric loss factor, the material would display higher EMI (SE). [START_REF] Chen | Microwave Electronics Measurement and Materials Characterisation[END_REF] In order to verify the dielectric properties of nanopapers at X band, PPy/ CNF (1:1) nanopaper was studied at a frequency of 10 and 15 GHz. The variation of dielectric constant (ε 1 ) and dielectric loss (ε 11 ) properties of PPy: CNF (1:1) composite paper at a frequency of 10 and 15 GHz is shown in Figure 9. At a frequency of 10 GHz, the nanopaper exhibited the dielectric properties of ε 1 = 2.93 and ε 11 = 0.96, whereas the dielectric values decreased as ε 1 = 2.15 and ε 11 = 0.93 at a frequency of 15 GHz. According to Maxwell-Wagner-Sillars (MWS) theory, ε 1 originates due to the matrix-filler mismatch at the interface of a polymer composites which outcomes the interfacial polarization. The decrease of ε 1 with increase in frequency for PPy/CNF nanopaper would be attributed to the charge dissipation at the matrix-filler interface. [START_REF] Pottathara | [END_REF] That means, at higher frequencies such as 15 GHz, the dipoles might attain the ability to violate the filed disparities and thus the charge accumulation will be lower. The excellent dielectric performance of PPy-CNF nanopaper in the high-frequency region makes it as a suitable candidate for electromagnetic shielding in the X-band region.

| Electromagnetic attenuation performance of fabricated PPy/CNF nanopapers

The electromagnetic screening ability of the fabricated PPy/CNF nanopapers was analyzed by recording the scattering parameters using a vector network analyzer (VNA) with an X band (8.2-12.4 GHz) waveguide. The SE value is usually expressed in terms of decibels (dB) which is the logarithmic ratio of the incident EM power to the transmitted EM power. The main criterion for achieving higher EMI SE value is to enhance the electrical conductivity of the system. 53 The formation of a well-defined coating of polypyrrole onto CNFs resulted in a higher DC electrical conductivity of 0.21 S/m for 1:1 PPy/CNF nanopapers. As per the existing EMI shielding theories, total SE (SE Total ) value of any EMI shield is the summation of three shielding components viz shielding by

Usually, when SE A component is higher than 10 dB, the SE MR value can be neglected and SE Total is the summation of only SE A & SE R .

The shielding effectivenees SE Total , SE A and SE R can be re-expressed in terms of scattering parameters as given below.

SE Total dB

ð Þ= 10 log 1 S 21 j j 2 = 10 log

1 S 12 j j 2 , ð2Þ
SE A dB ð Þ= 10 log 10 1 -S 11 ð Þ S 12 2 2 " # , ð3Þ
SE R dB ð Þ= 10 log 10 1 1 -S 11 ð Þ 2 " # , ð4Þ
Where, S 11 , S 21 , and S 12 are the scattering parameters could be traced from the VNA.

In the present study, 1 mm thick PPy/CNF nanopapers were used for all EMI shielding analysis as it was found to be optimum thickness from our previous work on Polyaniline (PANI)/CNF nanopapers. 42 Figure 10 shows the variation of EMI SE values of 1:0.2 and 1:0.5 PPy/CNF nanopapers with a maximum of ca. -8 dB and ca. -14 dB respectively at 8.2 GHz in X-band region with 1 mm paper thickness. However, as the concentration of PPy was further increased, it resulted in a higher EMI SE value of ca. -22 dB (>99% shielding) in Xband region as seen from Figure 11(a). This can be attributed to the higher electrical conductivity of the 1:1 PPy/CNF nanopapers, which has resulted in a higher EMI SE value. It is also noteworthy that all the PPy/CNF nanopapers suppressed the incoming electromagnetic radiations via a dominant absorption mechanism (ca. 89%) and minimal shielding by reflection (ca. 11%) (see Figure 11(b). The formation of strong intra molecular hydrogen bonding between the nitrogen of the polypyrrole and the hydroxyl groups of CNFs resulted in a uniform coating of polypyrrole on CNFs. These secondary forces are attributed to the strong affinity of polypyrrole on CNFs surface and thereby enhancing the EMI shielding ability of PPy/CNF nanopapers. These attributes make PPy/CNF nanopapers as a platform for developing and designing green EMI shielding materials for various end applications in radar and terrestrial communication systems.

For any EMI shielding material for practical device applications, a minimum EMI SE value of -20 dB is considered suitable for the smooth functioning of the electronic device. [START_REF] Pai | Nanocellulose Based Composites for Electronics[END_REF][START_REF] Pai | [END_REF] However, there are only limited reports on PPy/CNF based nanopapers as efficient microwave absorbers. Recently, L. Tang et al. (2015) fabricated flexible and highly conductive membranes via in situ polymerization of pyrrole monomer onto bacterial cellulose membranes. 56 These membranes exhibited a maximum EMI SE value of only -15 dB in the region of 500-3000 MHz. But, in this study, we enhanced the EMI (SE) value of PPy/CNF nanopapers to ca. -22 dB in X band (8.2-12.4 GHz) region which enables them applicable for commercial device applications. Table 2 lists out some of the existing works reported on EMI Shielding materials based on cellulose and one can infer that achieving an EMI (SE) value of ≥20 dB in cellulosic materials is a notable value so as to make these green EMI shields commercially viable.

In order to study the application of these nanopapers for EMI shielding application, the 1:1 PPy/CNF nanopapers were tested using an RF detector circuit and mobile phone. It was found that the RF detector could easily detect excess radiations emitted out from mobile phones when placed in close proximity to the device. A red led gives an indication of the excess radiation emitted as shown in Figure 12(a). When we place 1:1 PPy/CNF nanopapers in between the mobile phone and the device (see Figure 12(b)), the led does not glow which infers that the excess radiations are being absorbed by the fabricated nanopapers (see Video S2). The prime shielding mechanism of PPy/CNF nanopapers is via strong absorption of the incoming electromagnetic radiations and Figure 13 shows a cartoon for possible shielding mechanism of the fabricated nanopapers.

| Evaluation of skin depth

The vital thickness at which the intensity of the incident EM wave reduces to 1/e or 37% of its initial intensity is termed as skin depth of an electromagnetic shield. The skin effect is very much correlated with the shielding ability and it is desired to have a minimum skin depth for enhanced performance of the shields. 57 It can be expressed as given below.

Skin Depth

δ ð Þ = 1 ffiffiffiffiffiffiffiffiffiffi πf μσ p = -8:68 t SE A : ð5Þ
From the above equation, it can be observed that skin depth is inversely proportional to electrical conductivity. Hence, in the present study, PPy/CNF nanopapers with low electrical conductivity exhibited higher skin depth and vice versa. Nevertheless, all PPy/CNF nanopapers exhibited an effective attenuation by an absorption dominant mechanism, which will significantly reduce the skin depth of the nanopapers. The variation of skin depth of 1:1, 1:0.5 & 1:0.2 CNF:PPy nanopapers is shown in Figure 14, and was found to 0.41, 0.6, and 1.24 mm, respectively.

| CONCLUSION

The present study reports a facile approach for the fabrication of highly processable PPy/CNF suspensions containing various pyrrole ratios. The PPy/CNF flexible composite papers from an equimolar proportion of PPy/CNF exhibited a high-DC conductivity value of 0.21 S/cm. SEM images and FTIR studies revealed that a continuous network of PPy was formed on the surface of the CNFs as a result of the secondary interaction via hydrogen bonding between the former and the latter and is of substantial importance for attenuating the EM waves. The proposed composite nanopapers demonstrated a predominant absorption mechanism (ca. 89%) which was superior to reflection mechanism (ca. 11%) and were found to have a SE of ca. -22 dB for a 1.0 mm thick paper at 8.2 GHz. Also, these flexible nanopapers made by simple and easily scalable process are ideal for a good deal of applications such as flexible electrodes, sensors, and electrically conductive and flexible films and other flexible paper-based devices. Moreover, results from the EMI shielding measurements also confirmed that the PPy/CNF flexible composite paper is an excellent candidate for attenuating electromagnetic radiations. We envision that this study will open up a new platform for developing green microwave attenuators for future highend applications.

  FTIR absorption spectrum of PPy powder, PPy/CNF, and CNF nanopapers are given in Figure 4. From the F I G U R E 1 Pictorial representation showing the fabrication of PPy/CNF nanopaper via in situ polymerization of pyrrole monomers on cellulose nanofibers (CNFs) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4 ,

 4 Figure 4, neat CNF exhibited the absorption peak at 3305, 2902, 1430, 1371, 1058, and 897 cm -1 , which was due to the OH stretching, CH stretching, CH 2 andOCH in-plane bending, CH bending, C O C stretching, respectively.41,42 In the case of PPy, strong bands arising at 3218 and 1546 cm -1 was attributed to the N H stretching vibration and C C stretching of aromatic ring of PPy. Moreover, the peaks at 1306 and 1151 cm -1 corresponds to C H in-plane ring bending and C N in-plane ring deformation respectively.43 In the case of PPy/CNF nanopaper, a superposition of the characteristic peaks of both CNF and PPy was observed without any formation of new peaks. This infers that, there was no chemical reaction between both CNF and PPy. It is worth mentioning that the major peaks of PPy (3218, 1546, and 1157 cm -1 ) was shifted to lower wave numbers in PPy/CNF nanopapers (3206, 1544, and 1151 cm -1 ) which is ascribed to the strong

F

  I G U R E 2 Schematic representation for in situ polymerization of pyrrole onto CNF. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 3 Secondary interactions between CNF and PPy and digital photo of PPy/CNF paper. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 4 FTIR spectra of PPy/CNF and CNF flexible papers. CNF, cellulose nanofiber; FTIR, Fourier transform infrared [Color figure can be viewed at wileyonlinelibrary.com]

  (a)), which clearly signifying a good bonding between CNFs and PPy.

F 3 . 4 |

 34 I G U R E 6 SEM micrographs of (a) CNF paper and (b) PPy/ CNF paper. CNF, cellulose nanofiber; SEM, scanning electron microscope F I G U R E 7 SEM micrographs of the cross-section of (a) PPy/ CNF and (b) CNF paper. CNF, cellulose nanofiber; SEM, scanning electron microscope F I G U R E 5 XRD profiles of PPy powder, PPy/CNF and CNF nanopapers. CNF, cellulose nanofiber; XRD, X-ray diffraction [Color figure can be viewed at wileyonlinelibrary.com] Conductivity studies of the fabricated PPy/CNF paper

  (a) illustrates the image of the flexible PPy/CNF nanopaper, and Figure 8(b) shows the conductivity of the fabricated PPy/CNF (1:1) nanopaper (see Video S1).

F

  I G U R E 9 Variation of dielectric constant (ε 1 ) and dielectric loss (ε 11 ) of PPy:CNF (1:1) flexible nanopaper as a function of frequency at 10 and 15 GHz. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 1 0 EMI shielding effectiveness of the (a) (1:0.2) and (b) (1:0.5) PPy/CNF flexible paper at 1 mm thickness in the X band (8.2-12 GHz). CNF, cellulose nanofiber; EMI, electromagnetic interference [Color figure can be viewed at wileyonlinelibrary.com]

F

  I G U R E 1 1 EMI shielding effectiveness of the (1:1) PPy/CNF flexible paper at 1 mm thickness in the X band (8.2-12 GHz). CNF, cellulose nanofiber; EMI, electromagnetic interference [Color figure can be viewed at wileyonlinelibrary.com]

F

  I G U R E 1 2 (a) Excess radiation emitted from mobile phone detected using a RF detector circuit. (b) PPy/CNF (1:1) nanopapers absorbing the excess radiation from the mobile phone. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 1 3 Pictorial representation of EMI shielding mechanism of fabricated PPy/CNF flexible paper. CNF, cellulose nanofiber; EMI, electromagnetic interference [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 1 4 The skin depth v/s frequency plot for 1:0.2, 1:0.5, and 1:1 CNF:PPy nanopapers in the X band. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com]

  SE A ), shielding by reflection (SE R ), and shielding due to multiple internal reflections (SE MR ) SE Total dB ð Þ= SE A + SE R + SE MR :

	T A B L E 1 Conductivity values of PPy/CNF nanopapers with
	different PPy ratios
		DC conductivity
	CNF/PPy ratios	(S/cm)
	1:1.0	0.21
	1:0.9	0.02
	1:0.8	0.0089
	1:0.5	0.006
	1:0.3	0.0033
	1:0.2	0.00012

Abbreviation: CNF, cellulose nanofiber. F I G U R E 8 Digital images of (a) fabricated PPy/CNF paper (b) conductivity demonstration of PPy/CNF paper. CNF, cellulose nanofiber [Color figure can be viewed at wileyonlinelibrary.com] absorption (

  Comparison of shielding effectiveness of various cellulose-based nanocomposites

	Sr no	Polymer composites	Thickness (mm)	Frequency (GHz)	EMI SE (dB)	References
	1	(MWCNT)/cellulose	0.17	0.5-1.0	-20.3	58
	2	Bacterial cellulose/PANI membrane	0.080	8.2-12.4	-5	5 9
	3	Bacterial cellulose/PPy membrane		0.5-3.0	-15	26
	4.	cellulose/(CNT) composite paper	0.45	15-40	-20	60
	5	cellulose/CNT aerogel	2.5	8.2-12.4	-20	61
	6	CNF/PANI paper	1.0	8.2-12.4	-23	42
	7	CNF/CF paper	0.167	0.5-1.0	24.6	62
	8	PPy/CNF paper	1.0	8.2-12.4	-22	This study

T A B L E 2

Abbreviations: CNF, cellulose nanofiber; CNT, carbon nanotube; EMI, electromagnetic interference; MWCNT, Multi-walled carbon nanotube.

ACKNOWLEDGMENT Deepu A. Gopakumar is grateful to IMT Mines Albi, RAPSODEE CNRS, for providing Post-Doctoral fellowship. Avinash R. Pai would like to acknowledge the financial support received from Visvesvaraya PhD scheme for Electronics and IT, Digital India Corporation, (Formerly Media Lab Asia), MeitY, Government of India (Ref No: PhD MLA/4 (58)/2015-16, Unique Awardee No: MEITY-PHD-503). The authors gratefully acknowledge support from Université de Toulouse, IMT Mines Albi, RAPSODEE CNRS UMR, France, International and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, India and Federal University of Uberlandia, Brazil. Also, Thanks to National Council for Scientific and Technological Development-CNPq, Brazil (Process 421974/2018-4). ORCID Deepu A. Gopakumar https://orcid.org/0000-0001-9394-6302

Funding information

National Council for Scientific and Technological Development-CNPq, Brazil, Grant/Award Number: 421974/2018-4;

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of this article.


Delvallée Clarisse

Nicaise Samuel

Antin Manuela

Anne-Sophie Leuvrey

Nourisson Elsa

Leitch C Carmen

Kellaris Georgios

Stoetzel Corinne

Geoffroy Véronique

Scheidecker Sophie

Keren Boris

Depienne Christel

Klar Joakim

Dahl Niklas

Deleuze Jean-François

Génin Emmanuelle

Redon Richard

Demurger Florence

Devriendt Koenraad

Mathieu-Dramard Michèle

Poitou-Bernert Christine

Odent Sylvie

Katsanis Nicholas

Mandel Jean-Louis

Davis E Erica

Dollfus Hélène

Jean Muller
email: jeanmuller@unistra.fr

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet-Biedl syndrome Running title: Mobile element insertion in BBS1

Keywords: Bardet-Biedl syndrome, BBS1, Mobile element insertion, SVA F, Founder effect

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid-specific SINE-R/VNTR/Alu type F (SVA F) insertion in exon 13 of BBS1 in 8 families. In six families, the repeat insertion was found in trans with c.1169T>G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10,800 control individuals from gnomAD-SV) and due to a founder effect. Its 2,435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient-derived cell lines confirmed that the BBS1 SVA-F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation.

INTRODUCTION

Bardet-Biedl syndrome (BBS, MIM# 209900) is a rare autosomal ciliopathy associating retinitis pigmentosa, polydactyly, obesity, cognitive impairment and renal dysfunction. Its prevalence ranged from 1/160,000 live births [START_REF] Waters | Ciliopathies: an expanding disease spectrum[END_REF] to <1/50,000 in consanguineous or isolated populations [START_REF] Gouronc | High prevalence of Bardet-Biedl syndrome in La Réunion Island is due to a founder variant in ARL6/BBS3[END_REF] . Recent studies have delineated the antenatal and postnatal presentation of BBS patients [START_REF] Mary | Bardet-Biedl syndrome: Antenatal presentation of forty-five fetuses with biallelic pathogenic variants in known Bardet-Biedl syndrome genes[END_REF][START_REF] Niederlova | Meta-analysis of genotype-phenotype associations in Bardet-Biedl Syndrome uncovers differences among causative genes[END_REF] with pathogenic variants in 24 genes accounting for the molecular basis of a majority of cases [START_REF] Shamseldin | The morbid genome of ciliopathies: an update[END_REF] . Among the known BBS genes, BBS1 contributes the highest fraction of BBS patients (~20%) [START_REF] Khan | Genetics of Human Bardet-Biedl Syndrome, an Updates[END_REF] thanks to a founder variant (c.1169T>G, p.Met390Arg) [START_REF] Mykytyn | Evaluation of Complex Inheritance Involving the Most Common Bardet-Biedl Syndrome Locus (BBS1)[END_REF] . Among the unresolved cases in our cohort, several patients were heterozygous carriers of this variant suggesting two hypothesis: (1) a missed second primary driver allele in BBS1; or (2) participation of this variant as a second-site modifier in the presence of a different primary BBS driver locus [START_REF] Beales | Genetic Interaction of BBS1 Mutations with Alleles at Other BBS Loci Can Result in Non-Mendelian Bardet-Biedl Syndrome[END_REF][START_REF] Kousi | Evidence for secondary-variant genetic burden and non-random distribution across biological modules in a recessive ciliopathy[END_REF] . Notably, due to the high frequency of this variant in the general population (0.27% in non-Finnish Europeans; gnomAD) this variant could be detected coincidently. In this study, we report the identification of a mobile element insertion of SVA F type in 8 families, a portion of which are in trans to the recurrent c.1169T>G in BBS1 (n=6), and a subset as secondary sites in addition to a known recessive BBS locus (n=2). Already described as a single case report [START_REF] Tavares | Retrotransposon insertion as a novel mutational event in Bardet-Biedl syndrome[END_REF] , here we estimate the frequency of this variant in BBS (131 families) identifying this variant as the second most common pathogenic variant in BBS1. We also determined its full-length sequence using WGS and performed functional analysis to understand its consequences. In our cohorts, the insertion always occurs at the same genomic location and its recurrence in BBS populations is the result of a founder effect.

SUBJECTS AND METHODS

Subjects

Written informed consent was obtained from each participant and/or parent or legal guardian. The study protocols were approved by local Institutional Review Boards: "Comité de Protection des Personnes" (EST IV, N°DC-20142222), or the Lurie Children's Hospital IRB (IRB 2019-3057 or IRB 2019-2950). Our research complies with the Declaration of Helsinki. All patients were diagnosed with BBS according to Beales' clinical diagnostic criteria [START_REF] Beales | New criteria for improved diagnosis of Bardet-Biedl syndrome: results of a population survey[END_REF] (Supplementary Table 1). Patients (n=564) have been assembled into several cohorts (Supplementary Table 2).

Methods

All experimental procedures are reported as supporting information.

A c c e p t e d m a n u s c r i p t

RESULTS

Identification of an SVA F insertion in BBS1

According to our tiered diagnostic paradigm, we screened the recurrent BBS variants (BBS1:p.Met390Arg and BBS10:p.Cys91Leufs*5) in cohort 1 (n=217) and cohort 2 (n=192) followed by targeted exome sequencing (TES) in cohort 1. We identified seven individuals who carry heterozygous p.Met390Arg changes without a second pathogenic BBS1 variant identified in trans. In one family (family I), we identified a novel heterozygous deletion encompassing exons 4 to 11 (Supplementary Figure 1). Among the six p.Met390Arg variation-bearing cases, we leveraged a high depth of coverage to identify another putative pathogenic variant in exon 13 of BBS1 with the deletion/insertion of 2 bases, c.1215_1216delinsT (p.(Ala406Glnfs*4)) in family A.II-3 (Figure 1B and supplementary Figure 2) and 2 others (B.II-1, C.II-1). Careful analysis of the genomic region revealed a more complicated event leading to incorrect variant calling (Figure 1C). Indeed, soft clipped reads part (82 bp) revealed the insertion of an SVA F retrotransposon with a possible truncation of 311 bases 5' of the consensus sequence (Figure 1D). To screen additional patients not yet available by TES (cohort 2; 213 cases from 192 families), or an independent group with no pre-selection bias for BBS variations other than exclusion of BBS1 recessive cases (cohort 3; 134 cases from 126 families), we designed a duplex PCR including the SVA insertion point (Supplementary Figure 3). This led to the identification of 5 additional cases (family D to H) including 3 in trans to the p.Met390Arg and 2 as third or fourth allele (Figure 1A). Biallelic status of the variants and the exact same insertion point was confirmed for 7 individuals (Figure 1E). To estimate the overall incidence of the SVA F as a contributor of the BBS1 mutational burden, we explored all our BBS1 positive cases (e.g. biallelic pathogenic variants). Data from 131 unrelated cases of European ancestry corresponding to 262 pathogenic alleles in total (cohort 4) could be retrieved and account for 44 different pathogenic variations. Among those, the c.1169T>G represents 72.1% (189 alleles) while the second most frequent variant is the SVA F with a frequency of 2.3% (6 alleles).

Determination of the full length SVA F sequence

To understand the consequences of this insertion, we determined its full-length sequence. Given the repeated sequence of the SVA F, alignment of short reads onto the human genome generate artifacts (multimapped reads, split reads, discordant read pairs) that can be rescued using a dedicated bioinformatics pipeline. The initial TES aligned reads allowed us to identify 99 bp from the SVA F (Figure 2B). Assembled reads from WGS data (A.II-3) output a 387 bp contig composed of BBS1 (38 bp before the breakpoint in exon 13), a TSD (17 bp) and 330 bp that matched the SVA F consensus sequence. In conclusion, WGS data enabled accurate characterization of an additional 250 bp SVA F sequence (Figure 2). Based on this and the consensus SVA F sequence [START_REF] Kwon | Structure and Expression Analyses of SVA Elements in Relation to Functional Genes[END_REF] , we walked on the genome and determined an inserted sequence of 2,435 bp (Figure 2B and Supplementary Figure 4 and5). With the exception of a missing 311 bases (Figure 2A) in the 5' part, hallmarks of L1 mediated retrotransposition could be identified (Figure 2). The variant can now be described as c.1214_1215ins[MT113356], p.(Ala406Glnfs*47).

Functional impact of the SVA F insertion on BBS1

Predicted to create a premature stop codon, a reduced or absent mRNA expression due to nonsense-mediated decay potentially leading to a shorter protein (452 vs 593 AA) is likely to happen. Using primary cells from individual A.II-3, we demonstrated a significantly reduced BBS1 expression corresponding a priori to the loss of the SVA F allele (Figure 3A). However, a transcript carrying the SVA F is expressed (Supplementary Figure 6), albeit likely minor compared to the p.Met390Arg allele. Western blot analysis on protein extracted from A.II-3 cells revealed a single band corresponding to the normal BBS1 protein (Figure 3B) demonstrating the absence of a putative BBS1 polypeptide carrying the SVA F.

Founder effect or recurrent mechanism?

A c c e p t e d m a n u s c r i p t

Given the multiple carriers of this insertion in our cohorts (8 families), we wondered whether this MEI occurred as an independent (recurrent) mechanism or as the same mutational event derived from common ancestry (founder effect). First, no occurrence could be observed either from 1299 healthy individuals of European ancestry or from the 14,891 gnomAD SV controls [START_REF] Collins | A structural variation reference for medical and population genetics[END_REF] . Second, genotyping of 4 different families with SVA F alleles, along with 3 patients carrying the c.1169T>G change in the homozygous state (Supplementary Table 5) revealed the common c.1169T>G haplotype (~1.614 Mb) as well an SVA F allele of a much smaller size (~56.6kb) (Supplementary Table 5). PCA analysis confirmed the geographical distribution of the patients (Supplementary Figure 7). Haplotypes shared by the four SVA F carriers were compared to find the position where recombination events are likely to have happened. The estimation of the most recent common ancestor age was 74 generations 95% CI [33;190]. If we assume that one generation lasts 25 years, this will indicate an age of 1850 years 95% CI [825;4750]. Overall, this MEI is an ultra-rare event that arose from a common ancestor on one of the most common haplotypes at this locus and more recently than the Met390Arg haplotype.

A c c e p t e d m a n u s c r i p t

DISCUSSION

Although the diagnostic yield in BBS is relatively high, (60-100%), a substantial proportion of cases remain unresolved [START_REF] Mary | Bardet-Biedl syndrome: Antenatal presentation of forty-five fetuses with biallelic pathogenic variants in known Bardet-Biedl syndrome genes[END_REF][START_REF] Shamseldin | The morbid genome of ciliopathies: an update[END_REF] . This could be related to the attributes of the cohort (sample size and geographic origin), the clinical inclusion criteria (strict or more inclusive) or the genetic test used (TES, WES, WGS). In addition to the first BBS case with MEI reported by our group [START_REF] Redin | Targeted high-throughput sequencing for diagnosis of genetically heterogeneous diseases: efficient mutation detection in Bardet-Biedl and Alström syndromes[END_REF] and the recent SVA F description [START_REF] Tavares | Retrotransposon insertion as a novel mutational event in Bardet-Biedl syndrome[END_REF] , this is the largest description of such cases in BBS patients. Although rarely described, a number of MEI cases (>100) have been reported in rare diseases [START_REF] Taşkesen | Novel Alu retrotransposon insertion leading to Alström syndrome[END_REF] or in cancer [START_REF] Crivelli | Insertion of Alu elements at a PTEN hotspot in Cowden syndrome[END_REF] . The majority are Alu insertions [START_REF] Hancks | Active human retrotransposons: variation and disease[END_REF] and only a few SVA (n=16). Recently, large studies have evaluated the impact of MEI in patients between 0.04% to 0.15% of the cases [START_REF] Gardner | Contribution of retrotransposition to developmental disorders[END_REF][START_REF] Torene | Mobile element insertion detection in 89,874 clinical exomes[END_REF] . BBS1 is often the target of structural variations [START_REF] Lindstrand | Copy-Number Variation Contributes to the Mutational Load of Bardet-Biedl Syndrome[END_REF] . Interestingly, the BBS1 locus is GC rich and has a high density of L1 and Alu insertions that might explain the SVA F insertion (Supplementary methods). Given the nature of the SVA F sequence, which contains highly repeated elements, GC rich content and a poly(A) tail, complete amplification was not easily performed. We thus used the breakpoints identified by the TES and WGS to walk across the patient's genome and determine a 2,435bp insertion in exon 13 of BBS1. This inserted sequence is identical to the one already described [START_REF] Tavares | Retrotransposon insertion as a novel mutational event in Bardet-Biedl syndrome[END_REF] . Unlike most of the SVA elements in the human genome (63%) [START_REF] Wang | SVA Elements: A Hominid-specific Retroposon Family[END_REF] , this inserted SVA F is incomplete lacking 311 bases in 5' and possibly as almost all SVA it is inactive [START_REF] Hancks | Active human retrotransposons: variation and disease[END_REF] . Notably, beside short read standard WGS we also performed linked-read sequencing (10x genomics) to see whether this technology could overcome any of the technical challenges encountered through WGS and analysis (Supplementary Figure 8). Consistent with other reports, we did not find any significant advantage and it is now discontinued [START_REF] Uguen | Genome sequencing in cytogenetics: Comparison of short-read and linked-read approaches for germline structural variant detection and characterization[END_REF] . Interestingly sequencing of the insertion point revealed the exact same sequence for all patients and the genotyping of some individuals revealed a common shared haplotype. All families carrying the SVA F are of European ancestry: 4 originated from the western part of France (family A, C, E and F), one from the northern part of France (family B) and one from Denmark and Sweden (family D). These observations were highly suggestive of a single founder genomic event that we confirmed and estimated its age close to 1850 years. In conclusion, we identified the recurrence of a specific SVA F element in exon 13 of BBS1 in multiple BBS families highlighting the importance of detecting such genetic events even by TES. We determined the full length sequence of the inserted SVA F, show its pathogenicity and demonstrated the founder effect. This rare allele is the second most frequent cause of BBS1 after the c.1169T>G variant. We have also developed a duplex PCR, offering a simple and inexpensive way to detect this variant prior to any other large genetic screen. Accordingly we have modified our BBS diagnostic strategy (Supplementary Figure 10). (C) Magnified view of the incorrectly called deletion insertion with the sorting and coloring of the reads according to the "insert size". The sequence reveals all reads ending at the exact same position with a "AG" deletion and a "T" insertion (c.1215_1216delinsT). (D) Region of interest with the "Show soft-clipped bases" turned off revealing multiple reads with aberrant alignments on their right side and corresponding to an SVA F insertion. The spacing between the reads on the left (including the 3' end of the SVA F with the poly(A) tail) and the reads on the right (including the 5' end of the SVA F) corresponds to the TSD. (E) Segregation analysis using Sanger sequencing for M1 (left) and M2 (right).

Figure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. Schematic of the inserted SVA F element in exon 13 of BBS1. (A) Canonical SVA F structure composed of 6 parts: (1) hexameric CCCTCT repeats (2) Alu-like domain, (3) a Variable Number Tandem Repeats (VNTR) domain, (4) a Short Interspersed Nucleotide Elements (SINE-R) domain, (5) a poly(A) tail and (6) the target site duplication (TSD) at both extremities. Numbers above the scheme indicate the size of each element. (B) Schematic of both BBS1 alleles in A.II-3 with primer positioning and amplification products highlighting each contribution to the resolution of the full-length sequence.

Figure 3 .

 3 Figure 3. Quantification of BBS1 mRNA and protein levels in A.II-3 (A) Reduced BBS1 mRNA expression by RT-qPCR (A.II-3) compared to control or homozygous p.Met390Arg patient. The major fraction of BBS1 expression likely corresponds to the p.Met390Arg allele. Normalization was performed using both GAPDH and HPRT as reference genes. Mean of both results are shown. Controls are in gray (n=3), homozygous p.Met390Arg in black (n=2), and patient A.II-3 (p.Met390Arg and SVA F) in white (n=2). Bars show the mean of 2 independent experiments +/-SEM (n=2, t-test *: p<0.05, ns: not significant). (B) Western blot analysis of BBS1 protein level. No truncated version of BBS1 could be detected. Half reduced BBS1 protein level in A.II-3 compared to control. Bars show mean of 3 independent experiments +/-SEM (n=3, ttest *: p<0.05, ns: not significant). M: molecular weight ladder (kDa).

A

  c c e p t e d m a n u s c r i p t

ACKNOWLEDGEMENTS

We thank the patients and their family members for their participation, and the French Bardet-Biedl association for their support. Part of TES was performed by the Plateforme GenomEast (IGBMC, Illkirch-Graffenstaden, France) and for this, we thank Serge Vicaire, Murielle Philipps and Bernard Jost. We would like to thank Amélie Piton for scientific discussions and acknowledge Pierre Lindenbaum and every member of the FranceGenRef consortium.

FUNDING INFORMATION

This work has been published under the framework of the IdEX Unistra and benefits from a funding from the state managed by the French National Research Agency as part of the investments for the future program (JM). This work was supported by US National Institutes of Health grants HD042601 (NK), GM121317 (NK), DK072301 (NK and EED). NK is a Distinguished Valerie and George D. Kennedy Professor. WGS were supported by Fondation APLM and CREGEMES. CD is supported by an FRM grant (ECO20170637509).

data. The raw sequencing data generated in the course of this study are not publicly available due to the protocol and the corresponding consents used that did