dataesr/scientific-paragraphs-categorization huggingface dataset:
2014; O'Leary et al. 2015) , UNITE  (Kõljalg et al. 2013; Nilsson et al. 2018 ) and ISHAM-ITS  (Irinyi et al. 2015) , by selecting high-quality ITS sequences generated from reliably identified and preserved specimens. These reference databases play a critical role in the sequence-based species identification of herbarium specimens, cultured strains and fungal communities from environmental samples  (Tedersoo and Nilsson 2016). However, linking sequences to names remains a challenge, partly because of the incompleteness of these reference datasets  (Orock et al. 2012; Kõljalg et al. 2013; Crous et al. 2014; Nilsson et al. 2018). The molecular barcoding of herbarium specimens, including generic and species types therefore remains a priority  (Crous et al. 2014; Yahr et al. 2016). Although the characterisation of fungal diversity in environmental samples has benefited immensely from the development of next generation sequencing (NGS) technologies, the high-throughput generation of reference ITS sequences from herbarium specimens has been hindered by the short length of Illumina reads, the most commonly used NGS platform.
With a size ranging from 500 up to 1200 bp, the full length of the ITS region cannot be sequenced as a single DNA fragment. For DNA extractions of lichen specimens, which harbor on their surface or within their thalli a plethora of other fungi, as well as for any other DNA samples of mixed fungal communities, assembling DNA markers from a pool of short reads belonging to several species carries a high risk of obtaining chimeric sequences  (Hebert et al. 2018 ). As a consequence, fungal metabarcoding studies mostly use only half of the ITS region, either ITS1 or ITS2  (Nilsson et al. 2010; Mello et al. 2011; Blaalid et al. 2013). The generation of full length and high-quality barcodes from lichen specimens for reference databases or identification purposes generally involved Sanger sequencing (e.
For community analyses, CCSs can then be analysed by various software or software packages, such as Mothur  (Schloss et al. 2009)  or PipeCraft  (Anslan et al. 2017) , in order to demultiplex, filter, cluster and assign consensus sequences to a taxonomic unit (see  Anslan et al. 2018). Although PacBio (Pacific Biosciences) provides secondary analysis modules as part of the SMRT portal, none of these is specifically designed for community analysis. The Long Amplicon Analysis (LAA) pipeline available on SMRT Link and SMRT Portal attracted our interest, as it identifies differing clusters of sequencing reads within a single library and is capable of differentiating between underlying sequences that are 99. 9% similar, such as haplotypes and pseudogenes  (Bowman et al. 2014). The main application of LAA is allele phasing and detection in diploid organisms  (Bowman et al.
For each index/sample barcode, the output includes all unique consensus sequences obtained, together with their coverage value and predicted accuracies. LAA can be performed on the instrument's SMRT portal, allowing the sequencing provider to send these demultiplexed consensus sequences directly to the customer, saving time and effort in software installation and raw data analyses. In this study, the use of SMRT sequencing and the LAA pipeline for the production of ITS barcode sequences from lichen herbarium specimens was explored. The goals were: 1) to establish a high-throughput protocol to obtain indexed PCR products from lichen DNA extracts for SMRT sequencing; and 2) to investigate the ability of LAA to recover high-quality sequences for the target lichenised fungal species as well as for non-target fungal species.
Amplification, normalisation and pooling Indexed PCR products were generated using the PacBio Barcoded Universal Primers protocol (https://www. pacb. com/wp-content/uploads/2015/09/Procedure-and-Checklist-Preparing-SMRTbell-Libraries-PacB-Barcoded-Universal-Primers. pdf ). The ITS barcode (internal transcribed spacer 1, 5. 8S ribosomal RNA subunit and internal transcribed spacer 2) was the region targeted. With a first PCR, our target region was amplified using the primers ITS1F  (Gardes and Bruns 1993)  and ITS4  (White et al. 1990 ), both modified by adding a 5' block and a tail representing the PacBio universal sequences. In a 25 µl reaction, 5 µl of buffer, 1 µl of MyFi (Bioline, London, UK), 1 µl of 5 µM of each primer, 16 µl of water and 1 µl of DNA template (1/10 dilution) were added.
Library preparation, sequencing and SMRT Portal primary analysis The pooled sample (1. 5 µg of DNA) was sent to the Ramaciotti Centre for Genomics (UNSW Sydney, Australia) for single molecular real-time (SMRT) sequencing. The sample met the quality control requirements and showed two clear peaks at 650 and 923 bp, which are within the expected size range for the ITS barcode. The library preparation was done using the PacBio Barcoded Universal Primers protocol (https:// www. pacb. com/wp-content/uploads/2015/09/Procedure-and-Checklist-Preparing-SMRTbell-Libraries-PacB-Barcoded-Universal-Primers. pdf ). The sample was sequenced in one SMRT cell on a PacBio RSII using a P6 chemistry with a four-hour movie. The raw data were analysed using Long Amplicon Analysis (LAA v. 1) with barcoding option on the SMRT Portal.
Molecular identification and validation After demultiplexing, quality control and generation of consensus sequences using LAA, the amplicon data was provided in 96 files in fasta format. Sequences from the targeted lichenised fungal species were identified from fungal endophytes or contaminants based on sequence similarity using a BLASTN v 2. 8. 1 search on the nr database by excluding uncultured/environmental sample sequences (https://blast. ncbi. nlm. nih. gov/; search done on Dec 12-14 2018). The first BLASTN match (or highest bit score) was recorded unless it did not correspond to at least a fungal Class, in which case the next match was considered. For 12 samples, the target sequence obtained with PacBio was cross-validated by a sequence obtained with Sanger sequencing. The ITS barcode was amplified with the primers ITS1F and ITS4 using the DNA polymerase MyFi as previously described, and products were sent to Macrogen (Seoul, Korea) for purification and sequencing.
PacBio and Sanger sequences were manually compared in Mesquite v 3. 51  (Maddison and Maddison 2017). For samples for which multiple ITS barcodes were recovered for the target species, the sequence versions were compared using Mesquite and the BLASTN suite-2 sequences was used to estimate percentage identity between selected sequence pairs. Sequences with the highest coverage were selected as primary barcodes. Secondary barcodes were only taken into consideration when their coverage was above 30 and when they were less than 98% similar from the primary barcode. Raw PacBio files and FASTQ files of primary and secondary barcodes obtained were deposited in the Sequence Read Archive on NCBI (BioProject ID PRJNA541190).
Some reactions with weak products did generate a sequence for the target species, although generally with a coverage lower than 100 (e. g. , samples 29, 94 and 96). For 46 samples, a single sequence was obtained for the target species among all the co-amplified fungal taxa. For the other samples, the clustering and phasing process resulted in 2-9 sequences that could be attributed to the target species using the BLASTN match. The similarity between these different sequence versions was investigated in an alignment. After reverse complement and barcode trimming, these sequence versions were in fact identical for an additional ten samples, bringing the total of successful target single-sequences to 56. A few sequence versions also differed with indels in long single nucleotide repeats (e. g.
Discussion Collections of well-curated lichen specimens are important resources for species identification, whether done traditionally using morpho-anatomical together with chemical characters, or using DNA barcodes. For the latter, access to databases with high quality sequence data, detailed voucher information and reliable taxon names is critical. Although current reference sequence datasets exist, they are still incomplete. Thus, the molecular barcoding of lichen specimens for a broad range of species remains a priority  (Yahr et al. 2016). In this study, a SMRT sequencing metabarcoding approach generated ITS sequences for 85 of the 96 lichen specimens investigated. This study shows that for lichen herbarium specimens up to 25 years old, full length and highquality ITS sequences can be recovered for the target lichenised fungus using PacBio amplicon sequencing.
Furthermore, sequences were also obtained for co-amplifying fungi, shedding light on the diversity of endolichenic fungi in the samples. The protocol used in this study enabled the high-throughput processing of samples and the bioinformatic pipeline permitted the recovery of high-quality sequences with minimum time and effort, because both the primary and secondary analyses could be carried out by the sequencing provider. For mixed lichen DNA extracts, SMRT sequencing is a cheaper option than cloning and Sanger sequencing when high number of samples need to be processed. The cost of producing a barcode Sanger sequence is generally around AUD$15/sample (including DNA extraction, amplification and sequencing costs), but when cloning is required, this cost can increase to AUD$100/ sample. With the PacBio amplicon protocol used in this study and the PacBio RSII platform, the cost per sample was estimated at AUD$37.
This cost could be further reduced by co-amplifying more markers and/or multiplexing more samples. With the new PacBio platform Sequel, multiplexing 384 samples for one marker would bring the cost down to AUD$12/sample. Moreover, with the recent increased output per SMRT cell (from 1 gigabase for RSII to 20 gigabases for the new PacBio platform Sequel), the cost per sample could fall even further with higher multiplexing levels. The use of PacBio amplicon sequencing for high-throughput barcoding of lichen specimens is therefore very promising. Some challenges remain, both for the laboratory and the bioinformatic aspects of this method. Some considerations and suggestions for improvements based on this study are outlined below, especially with regard to the preparation of the amplicon library and the generation of ITS barcode sequences using the LAA pipeline.
In our preliminary test of this protocol, only a small number of PCR products could be recovered using the high-fidelity Phusion Hot Start II (Thermo Scientific). As the focus of our study was to obtain a barcode sequence from the target species, and not to characterise fungal communities using sequences, the polymerase MyFi was used to generate amplicons. It is marketed as being efficient with challenging templates and inhibitor-rich samples and has so far worked well for lichen specimens. Although not as accurate as a high-fidelity polymerase, MyFi comprises a proofreading component that allows a 3. 5× higher fidelity than Taq DNA polymerase. In addition to polymerase misincorporation, chimera formation is another common PCR-induced source of error. Low starting template concentration and low amplification cycle numbers tend to reduce the rate of chimera formation  (Lahr and Katz 2009).
2014). In this study, as an alternative to cloning, the LAA pipeline was used to recover high-quality barcode sequences from mixed DNA samples of target and nontarget fungal species occurring in lichen specimens. The LAA pipeline is accessible on the SMRT portal and can be run by the sequencing service provider together with the primary analysis. The output data generated by LAA included fasta and fastq files with all sequence variants, as well as accuracy and coverage values for each of these sequences. Sequences with the highest coverage generally corresponded to the target species and for more than half of the samples, one single sequence was recovered for the target species. For the other samples, 2-8 sequence versions were recovered per target species. Most often the ITS variation within sample was caused by low quality consensus sequences, which could then be identified due to their low coverage.
Acknowledgment The authors would like to thank  Judith Curnow  ( Australian National Botanic Gardens, Canberra ) for her help with specimen databasing and curation,  Michel Bertrand  and  Jean-Yves Monnat  for providing lichen specimens, and  Lan Li  ( Australian National Herbarium, Canberra ) for her help with the molecular work. We also thank  Tonia Russell  ( Ramaciotti Centre for Genomics, UNSW Sydney, Sydney ) and  Pacific Biosciences  for their advice with sample preparation and data analysis.
This study presents a new wireless measurement system for outdoor fire experiments based on an OCARI wireless sensor network (WSN). The open-source radio communication OCARI stack ('Open Communication Protocol for Ad Hoc Reliable Industrial Instrumentation') allows to overcome the limitations of the previous wireless solutions for fire measurements, especially those based on proprietary systems, such as Zigbee, wherein users cannot control energy consumption and timestamping. This paper presents the design of an Atmel/ARM-based platform compatible with the wireless OCARI stack, adapted to record and communicate data from heat transducers-namely, a K-type thermocouple and heat fluxmeter. A 5-node WSN using heat sensors faces a 10 m 2 natural fire of excelsior (pine wood) in outdoor conditions.
Implementation In a previous study on ZigBee devices for heat measurements in fire, we observed significant delays for the data gained on the WSN when compared with that for equivalent wired devices  [6]. We used an OCARI protocol stack to offer an appropriate quality of service to develop a fire metrology system for the field scale to overcome delays. The aim of this study involves determining if the two innovative elements of the OCARI stack (i. e. , MaCARI protocol and its OSERENA scheduling algorithm) when compared with that of the Zigbee one can provide the quality of service if used conjointly for fire measurements. As a consequence of new skills (i. e. , MACARI and OSERENA used conjointly), OCARI makes it possible to switch off the network nodes during a cyclic schedule, allowing a wake-up cycle, measurement cycle, and data transmission cycle.
Heat Measurement Devices Heat data are collected with standard thermocouples and radiative heat fluxmeters (HFMs) that are typically used for fire experiments  [1] [2] [3] [22] [23] [24]. We performed two types of heat measurements as follows: • Measurement of gas temperature (in both air and fuel beds): Gas temperature T is measured around the transducer and makes it possible to detect the presence of smoke (60 • C < T < 500 • C) or flame (500 • C < T ) as per fire safety engineering standards; • Measurement of radiant heat fluxes: The system measures heat radiation from the fire to the target sensor. The measurement of the gas phase temperature is performed with a K-type thermocouple that corresponds to a transducer that obeys the thermoelectric effect or Seebeck effect. This effect is due to a junction (hot weld) of two electrical wires wherein each is composed of a different alloy at T 1 temperature.
Wired System: Campbell CR3000 Datalogger On the wired network, the heat quantities-namely, temperature and radiant heat flux-are recorded using a portable CR3000 datalogger model (Campbell Scientific-Edmonton, AB, Canada c ). This is dedicated to work in outdoor conditions and was already used to collect data during large-scale fire spreading experiments  [2, 6, 22, 23]. It also serves as the reference wired datalogger to evaluate the response of the Zigbee WSN tested in Reference  [6]. For a fair comparison, we used the same wired datalogger with an identical setup. Specifically, on the CR3000, there are 14 differential analogue channel inputs. Each channel exhibits a maximum scan rate of 100 Hz. The recorded voltage levels are digitised with a 16-bit resolution. We selected five voltage levels ranging from ±50 mV to ±5 V.
In this study, the channels are configured at a ±50 mV (analogue resolution: 0. 67 µV per digit). We selected a sampling rate corresponding to 1 Hz. The data were stored in 4 MB of memory. It is necessary to bury the wires from the devices to the datalogger into the ground to protect them from the effects of the fire. Furthermore, a thermal insulation composed of an assembly of ceramic fibre protects the off-ground section of the datalogger. This protection is intended to make the wires resistant to the fire. The analogue data were encoded on 16 bits on the wired datalogger and timestamped at the instance of the digitisation. We estimated the delay for transmitting the information from the transducer to the datalogger for a cable with a length ranging to a few metres as approximately 9 ns based on the CR3000 datasheet.
OCARI-Based Network for Heat Measurements in Fire Experiments The wireless platform runs on the OCARI software prototype for wireless communication based on the Adwave ADWRF24-LRS technology (Figure  4 ). It generates a digitised data flow from the measurement area to the network coordinator (CPAN). Each OCARI network node consists of a radio communication device based on an Atmel microcontroller SAM3. Each radio device is coupled with an acquisition interface in which two analogue transducers are connected through a 12-bit ADC on which a real-time circuit (RCT) operates. The RCT is integrated to generate a timestamp at the moment of analogue-to-digital conversion. This type of a timer circuit device is energy-consuming, although the ability of OCARI stack to promote very low energy consumption solutions increases due to reasonable RTCs.
All plots exhibit a different time sampling between both wired and wireless data acquisitions with similar temperature signals. Specifically, the wired datalogger records digitised information from each thermocouple (TC) as sampled at 1 Hz. The wireless system works differently wherein it records data packaged in each message reaching the coordinator of the personal area network (CPAN) successfully. In each message, timestamping of the AD conversion exists at every note at the microcontroller level (Figure  5 ). Therefore, in each figure from   we observe data and the timestamp that was received by the CPAN from the WSN. This implies that the sampling of analogue signals on the WSN is observed a posteriori as the reconstruction of the couple timestamp-data in each validated message. Given the possible constant message losses in this type of a harsh environment (e.
Conclusions The study presented a solution to overcome the limitations of WSN for heat measurements during outdoor fires and specifically for the ones observed with the Zigbee communication protocol  [6]. The advantages of using an OCARI-WSN for this type of applications include cost compression due to wire suppression as the modularity and flexibility of the measurement system in an open-source WSN. During the detailed presentation of the WSN solution, sensors, and standard datalogger, we discussed the different opportunities ranging from cable heat transducers to voltage amplifiers for interfacing sensors with the WSN node. Subsequently, we evaluated the performance of the OCARI-based WSN for temperature and heat flux measurements. We compared the data recorded on the wireless system to the ones gained on a standard wired datalogger during a series of fire experiments in the open.
e. , performing long-term measurements because of its energy consumption. Let us recall as a conclusion that except Reference  [6] , the literature does not report on Zigbee-based WSN systems able to perform fire measurements. The list  [7] [8] [9] [10] [11]  refers merely to detection. quality of service from OCARI with respect to Zigbee and due to the MACARI implementation is the first feature of the new WSN. Given the aim of developing a wireless measurement system, it is necessary to ensure that the deviation of the local clocks of the nodes during the synchronisation process does not consequently delay time slicing and does not affect the measurement quality within the allocated time and specifically during fire experiments at the field scale. Given the OSERENA scheduling algorithm, the collecting experimental data system is equipped to resist disruption that may be created by large flames.
The OSERENA algorithm relies on the theoretical principles of graph colouring algorithms that are commonly used to enhance the effectiveness of MAC layer protocols. Generally, the algorithms consist of assigning a colour to graph vertices of a routing tree wherein the root is a data well  [30] [31] [32] [33]. The allocation of colours is performed such that two adjacent nodes do not exhibit the same colour, and thus does not emit in the same time slot. In the OSERENA algorithm, nodes coloration permits alternation over node activity and standby periods in which the radio module is switched off to save battery energy. A maximum 3-hop colouring is implemented for the node to schedule transmissions on the medium. Subsequently, during the neighbourhood detection phase, each node determines its neighbour nodes by periodically emitting "Hello" messages to its adjacent nodes at the 1-hop.
Together, these data identify the replacement of valine by tyrosine at RPS2 aa position 226 as a ram mutation and demonstrate that the transfected RPS2 is incorporated into actively translating ribosomes. Effects of RPS2 A226Y on translation. Cell viability was not measurably affected in the RPS2-A226Y mutants (Fig. 1b ), however, we detected a significantly longer generation time compared to RPS2 wt transfected cells (Fig. 1c ). This loss of fitness, a hallmark of ram mutations, was exacerbated by the aminoglycoside geneticin (Fig. 1c ). To assess whether A226Y-induced mistranslation resulted in increased protein aggregation in the cell, we used cells transfected with a misfolding-prone firefly luciferase reporter  25. Using antibodies against Fluc, we performed western blots of the soluble and pellet fractions of cellular extracts.
3d ). Translation and protein quality control pathways. We found decreased transcript levels of genes involved in cytosolic protein synthesis, including structural ribosomal proteins, aminoacyl tRNA synthetases, and elongation factors (Supplementary Fig. 4a, b ), consistent with our experimental data showing reduced translational activity and elongation slowdown. We also found increased transcript levels of ribosomal release factors eRF1 and eRF3 (Supplementary Fig. 4b ), most likely reflecting a response to A226Y-mediated increased stop codon read-through. In addition, we observed elevated transcript levels for cytosolic chaperones and proteasome components (Supplementary Fig. 4c, d ). Thus, A226Y-induced mistranslation upregulates cytosolic protein quality control pathways. We corroborated these findings by western blot analysis to detect ubiquitination at the protein level and by measuring the proteolytic activity of the 26S proteasome.
HEK293 cells were transfected with pRPS2 (WT/A226Y) expression vectors using Turbofect (Thermo Fisher Scientific) and cultured in DMEM with 10% FBS under 100 µg/ml hygromycin B selection for 5-7 weeks. GFP expressing colonies were picked for further characterization. GFP fluorescence was analyzed by flow cytometry using the BD FACS Canto II (BD Biosciences) and the FlowJo software (Tree Star Inc). Several independent clones from each pRps2-WT and pRps2-A226Y transfected cells were used for all further experiments; the number of independent clones used in each experiment is specified in the corresponding figure legend. Plasmids MTS-mCherry-GFP1-10, FlucWT-HA-GFP11, and FlucDM-HA-GFP11 were obtained from Addgene, USA  40. Neo R cassette in vectors FlucWT-HA-GFP11 and FlucDM-HA-GFP11 was replaced with Amp R and Hyg R cassettes from vector pGL4.