dataesr/scientific-paragraphs-categorization huggingface dataset:
Discussion In this study, we illustrate that a two-step PCR approach followed by Illumina sequencing can be used for integrative taxonomy or for increasing the number of animal or plant species and specimens in barcode databases. When nuclear genes are included in the set of target markers, this method can allow accurate identification of specimens within species complexes where mitochondrial markers may be misleading (because of intrinsic problems such as introgression  6, 7  ). Interestingly, primers and PCR conditions used to generate Sanger data sets did not need optimisation to be used for MiSeq library preparation. Moreover, this approach does not require costly investments in laboratory equipment and supplies. Provided that adapters/index and primers are compatible (e. g. no hairpin structure), researchers can keep on working with markers they have previously selected for informativeness  33  to resolve shallower phylogenetic relationships within their target group.
The use of mini-barcodes has been shown to be powerful enough to characterize metazoan diversity  36, 37. However, to increase identification success, having only complete COI sequences in reference databases seems more appropriate. In that sense, until read length is sufficient enough to allow sequencing of fragments over 600 bp, using two overlapping fragments such as the FC and BR fragments developed by Shokralla et al. 2  (large spectrum of taxa), our COI-long/COI-short fragments (for fig wasps), or overlapping fragments that are easier to amplify in the target group, would be more appropriate for the construction of reference databases. Our results suggest that it may be helpful to sequence PCR products with no visible bands on the gel (potentially negative PCR). Indeed, we obtained sequences that passed our quality control steps for 86 specimens for which no amplification product was detected on the gel.
At least a translation to amino acids and a comparison to existing database (e. g. through BLAST) should be performed before sequence validation. While it has been underlined that pseudogenes, heteroplasmic sequences or sequences from symbionts or parasites may be obtained  4  , contamination during library preparation is less discussed. Indeed, contamination is difficult to detect and requires taxonomic knowledge and sequencing of both mtDNA and nuDNA markers to be distinguished from mtDNA introgression. Our results confirm that several markers should be sequenced for species diversity assessment to avoid underestimation of the number of species. While it may be possible to identify cross-contamination or amplification of non-coding copies (e. g. nuclear mitochondrial pseudogenes, NUMTS), sorting out paralogs in which no stop codon or frameshift mutation is detected may be difficult.
To conclude, two-step PCR approaches should contribute to accelerating the global effort of improving the state of completeness and accuracy of present reference databases. This will be facilitated by the ease of switching from multigenic, Sanger-based methods to NGS methods, as presented here. We advocate capitalizing on the    Table 4. Final results obtained on the combined data set (Sanger + MiSeq), after completion of the workflow. 1 At least one of the two PCR reactions (COI-short or COI-long) was positive. 2 At least one of the two PCR reactions was positive for at least one specimen. 3 The two PCR reactions were negative. 4 The two PCR reactions were negative for all specimens. huge investments made to construct barcode databases (BOLD) and in practice be conservative and pragmatic in maintaining the genes and the methods mastered by scientists, while shifting to next generation sequencing.
Methods Study group. We used the fig wasp genus Ceratosolen (Hymenoptera: Chalcidoidea: Agaonidae) as a test case because it is a relatively diverse genus of insects (encompassing an estimate of 230 species worldwide, of which only 71 are described). This genus pollinates Ficus species of the subgenus Sycomorus (158 described species worldwide) and is thoroughly studied by researchers working on figs. The genome of one species of Ceratosolen has been recently sequenced  43. Furthermore, in the last 20 years, we have developed a multigenic barcoding database using Sanger sequencing that encompass an unprecedented sampling of species. We have previously sequenced these specimens on COI, Cytb and EF1α. Sampling. One-hundred twelve species of Ceratosolen were included in the data set, of which about half (n = 62) are undescribed.
Analyses of the MiSeq data set. Step 1, from read filtering to clustering. Quality control checks were performed on raw sequence data with FastQC v. 0. 11. 2  60  (Fig. 2 ). Overlapping paired-end reads were reassembled using FLASH v. 1. 2. 11  61  with default settings and extended maximum overlap length (300). When paired-end reads did not overlap (COI-long and EF1a, Table  1 ), forward and reverse reads were analysed separately. CUTADAPT v. 1. 2. 1  62  with default settings was used to sort paired reads by gene region and remove primers. COI-long and EF1a forward and reverse reads were quality trimmed (reads were truncated at the first position having quality score < 21) using VSEARCH v. 1. 8. 1 (available at https://github. com/torognes/vsearch). After removing primers and quality filtering, fastq files were converted to fasta files and sequences less than 150 bp in length were filtered out using VSEARCH.
Remaining sequences were dereplicated and putative chimeric sequences were removed using VSEARCH. Cytb and COI-short sequences were then trimmed by length using PRINSEQ v. 0. 20. 4  63  (minimum length = 400 bp; maximum length = 550 bp). Illumina sequences were then clustered using SWARM v. 2. 1. 6  64 and CAP3  65  with default settings. Finally, clusters containing less than 10 sequences were excluded from the data sets using VSEARCH. Difference in read lengths due to quality trimming leaded to an overestimation of the number of clusters by SWARM for COI-long and EF1a forward and reverse reads. Therefore only the results obtained with CAP3 were subsequently analysed.
Analyses of the MiSeq data set. Step 2, quality control of clusters of reads. For each gene region, the consensus sequence of each cluster was aligned with the corresponding Sanger data set using MAFFT v7. 222 (default parameter) (Fig. 3 ). When paired-end reads did not overlap (COI-long and EF1a), clusters of reads 1 and clusters of reads 2 were analysed separately. At this step of the process, 6 data sets were assembled:COI Sanger + COI-short MiSeq, COI Sanger + COI-long MiSeq reads 1, COI Sanger + COI-long MiSeq reads 2, Cytb Sanger + Cytb MiSeq, EF Sanger + EF reads 1 MiSeq, EF Sanger + EF reads 2 MiSeq. Alignments were translated to amino acids using Geneiousv6. 1. 6 to detect frameshift mutations and premature stop codons. Non-coding sequences were removed from the data set. NCBI-BLAST was used to identify sequences that did not belong to the target group.
Phylogenetic trees were then inferred for each gene region using RAxML. Resulting trees were visualised and annotated using TreeGraph 2  53. Visual inspection of trees was carried out to identify contaminations, which were subsequently removed from the data sets. Reads 1 and 2 for COI and EF were then merged into a single data set (gaps were inserted between the non overlapping reads). Finally, COI-long and COI-short data sets were merged using Geneious v6. 1. 6. When COI-long and COI-short sequences were exactly identical in their overlapping part, they were merged to get the longest possible COI sequence. When differences were observed, they were not merged and both sequences were kept in the final data set. MEGA7 66 was used to calculate average divergence (p-distance) between sequence groups for problematic cases.
Acknowledgements We thank  Maxime Galan  ( INRA, CBGP, France ) and  Adrien Vigneron  ( Newcastle University, UK ) for technical advises;  Laure Benoit  ( CIRAD, CBGP, France ) and  Guenaëlle Genson  ( INRA, CBGP, France ) for their help with lab work;  Hélène Vignes  ( CIRAD, GPTR, France ) for sequencing of the library as well as  Alexandre Dehne-Garcia  ( INRA, CBGP, France ) for his help with the CBGP HPC computational platform on which analyses were performed. We thank  Anthony Bain  ( Taipei University ),  Yang Da-Rong ,  Zuodong Wei  and  Peng Yan-Qiong  ( Xishuangbanna Botanical Garden, China ),  Rhett Harrison  ( CTFS, Malaysia ),  Rong Chien Lin  ( University Taipei, Taiwan ),  Emmanuelle Jousselin ,  Carole Kerdelhué  ( CBGP Montpellier, France ),  Marc Pignal  ( MNHN Paris, France ),  Etienne Randrianjohany  ( CNRE, Madagascar), Simon van Noort (Iziko Museums of South Africa ),  Rosichon Ubaidillah  ( LIPI Bogor, Indonesia ) for providing samples and two anonymous reviewers for their comments on this manuscript.
Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy.
Patients and samples selection Written informed consent for genetic analysis of tumor and plasma samples was obtained for all patients at Gustave Roussy Cancer Center for the MOSCATO 01 and MATCHR protocols. These protocols were approved by local Institutional Review Boards CSET 2011/1755 (NCT01566019 trial) and CSET 2014/2144 (NCT02517892 trial), as well as regional ethics committee CPP ILE DE FRANCE VII, and were conducted according to the Good Clinical Practice Guidelines of the International Conference on Harmonization and the Declaration of Helsinki recommendations. We selected cfDNA samples from thirty-two non-consecutive patients harboring advanced cancer, based on availability of remaining cfDNA material  [16] , as well as availability of cfDNA-TGS and tDNA-WES results (Fig  1 ). Corresponding tumor biopsies presented a range of TML values.
The supernatant containing the plasma was further centrifuged at 14,000 g for 10 minutes at room temperature and stored at 80˚C until analysis. DNA was extracted from 2 mL of plasma using QIAamp circulating nucleic acid kit (Qiagen), according to manufacturer's instructions, and suspended in 40 μL of AVE buffer. A real-time quantitative PCR Taq-Man™ assay targeting GAPDH was used to measure plasma DNA concentration. Whole exome sequencing (tDNA and cfDNA) Library preparation, exome capture, sequencing and data analysis were done by IntegraGen SA (Evry, France). Genomic DNA was captured using Agilent in-solution enrichment methodology (SureSelect SureSelect XT Clinical Research Exome, Agilent) with a biotinylated oligonucleotides probes library (SureSelect XT Clinical Research Exome-54 Mb, Agilent), followed by paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000  [18].
WES bioinformatics analysis (tDNA and cfDNA) Base calling was performed using the Real-Time Analysis software sequence pipeline (2) with default parameters. Sequence reads were mapped to the human genome build hg19 (GRCh37) using Elandv2e (Illumina, CASAVA1. 8. 2) allowing multiseeded and gapped alignments. Duplicated reads (e. g. paired-end reads in which the insert DNA showed identical start and end positions in the human genome) were removed. CASAVA1. 8. 2 was used to call single-nucleotide variants (SNVs) and short insertions/deletions (maximum size was 300 nucleotides), taking into account all reads per position. Indels with Q(Indel) < 20, or regions with low mappability (QVCutoff < 90) were filtered out. An in-house algorithm was used to compare normal and tumor genotypes from exome sequencing data in order to determine the somatic nature of the variation.
Sequencing was made using Ion Personal Genome Machine (PGM, Life Technologies). Sequencing data were analyzed with Torrent Suite Variant Caller 4. 2 software and mapped to hg19. Variants were called if over 5 reads supported the variant and/or total base depth >50 and/or variant frequency >1% was observed. All variants identified were visually controlled on. bam files using Alamut v2. 4. 2 software (Interactive Biosoftware). All germline variants found in 1000 Genomes Project or ESP (Exome Sequencing Project database) with frequency >0. 1% were removed. Filtered variants were reviewed, classified and annotated by an expert molecular medical geneticist according to available knowledge (COSMIC, TCGA and medical literature). Variants of interest were selected to generate clinical reports. All retained variants are reported here as "curated variants" or "mutations".
Comparison of cfDNA with two biopsy sites The added value of cfDNA-WES was evaluated for tumor heterogeneity assessment by comparing liquid biopsies to multiple matched tissue biopsy sites. WES analyses from two biopsy sites were available for four of the cfDNA samples (Fig  6 ). For 3 of them, both sites were synchronous with the plasma sample, whereas for P30, one biopsy was synchronous with the plasma sample, the other biopsy dated 5 months before. CfDNA-WES presented between 177 and 331 variants. Amongst them, between 9 and 49 were shared with a single biopsy site, while between 7 and 30 were shared with both biopsy sites. This data supports the hypothesis that liquid biopsy may capture the entire heterogeneity of the disease and enable tracking of subclonal dynamics  [19].
Acknowledgments The authors acknowledge the clinical team, including  Dr. Christophe Massard ,  Dr. Charles Ferte  ´,  Dr. Antoine Hollebecque ,  Dr. Loic Verlingue ,  Dr. Rastilav Bahleda ,  Dr. Anas Gazzah ,  Dr. Andrea Varga ,  Dr. Sophie Postel-Vinay ,  Dr. Yohann Loriot ,  Dr. Caroline Even ,  Dr. Thierry De Baere ,  Dr. Frederic Deschamps ,  Dr. Philippe Vielh ,  Dr. Jean-Yves Scoazec ,  Dr. Vincent Ribrag , for their implication in enrollment of patient in MOSCATO trial, and  Maud Ngo-Camus ,  Aljosa Celebic  for Clinical Data Management. The authors also acknowledge the laboratory and bioinformatic teams, including  Melanie Laporte ,  Isabelle Miran ,  Nelly Motte,  Catherine Richon , Aurelie Honore,  Gladwys Faucher ,  Zsofia Balogh ,  Jonathan Sabio ,  Lionel Fougeat ,  Marie Xiberras ,  Leslie Girard ,  Catherine Lapage ,  Guillaume Meurice ,  Yannick Boursin ,  Marc Deloger ,  Bastien Job , for their support in the preanalytic processing of samples, storage and for tumor sample analysis (wet lab and bioinformatic) of the patients included into MOSCATO 01 trial.
Statistical analysis All statistical calculations were performed using IBM SPSS Statistics (IBM SPSS Statistics for Windows, Version 22, SPSS Inc. an IBM Company) program and Microsoft Excel 2010 (Microsoft Corporation, Seattle, WA, United States). The descriptive analysis of the epidemiological data presented as frequencies, percentages and mean ± standard deviation (SD). χ 2 test was performed to find out the variables associated with cancer among the surgical patients.
The outline of this paper is as follows. In Section 2 we summarize the theoretical input, beginning with a description of the dominantly wino MSSM parameter region satisfying the relic-density constraint, then providing some details on the computation of the DM annihilation rates to primary two-body final states. The following Section 3 supplies information about the implementation of the constraints from diffuse gammarays from the dSphs, galactic CRs, direct detection and the CMB, and the data employed for the analysis. The results of the indirect detection analysis are presented in Section 4 as constraints in the plane of the two most relevant parameters of the MSSM, the wino mass parameter M 2 and |µ| -M 2 , where µ is the Higgsino mass parameter. In Section 5 the indirect detection constraints are combined with that from the non-observation of dark matter-nucleon scattering.
For primary electrons we use a broken power-law with spectral indices 1. 6/2. 65 and a break at 7 GV, while for heavier nuclei we assumed one power-law with index 2. 25. R i 0,1 refer to the positions of the first and second break, respectively, and γ i 1,2,3 to the power-law in the three regions separated by the two breaks. The propagation parameters were obtained by fitting to B/C, proton and He data and cross-checked with antiproton data, while the primary electrons were obtained from the measured electron flux. rameters are given in Table  1. In Fig. 3  we show the fit to the B/C and the AMS-02 proton data  [23] [24] [25]  and superimpose the older data from PAMELA  [26, 27]. In all these cases, as well as for the lepton data  [28, 29] , the measurements used in the fits were from AMS-02 results only.
Acknowledgements We thank  A. Ibarra  for comments on the manuscript, and  A. Goudelis  and  V. Rentala  for helpful discussions. This work is supported in part by the  Gottfried Wilhelm Leibniz programme of the Deutsche Forschungsgemeinschaft (DFG)  and the  Excellence Cluster "Origin and Structure of the Universe" at Technische Universität München. AH is supported by the  University of Oslo  through the  Strategic Dark Matter Initiative (SDI). We further gratefully acknowledge that part of this work was performed using the facilities of the  Computational Center for Particle and Astrophysics  ( C2PAP ) of the Excellence Cluster.
Recently, tivantinib has demonstrated improved progression-free and overall survival in a randomize phase 2 second line study in a subgroup of advanced HCC patients with high expression of MET whereas no clinical benefit was observed in the low MET-expressing group  (9). These encouraging results led to the development of the first biomarker-based phase 3 clinical trial in HCC and there are currently, two phase 3 ongoing clinical trials (NCT01755767, METIV-HCC; NCT02029157, JET-HCC) evaluating tivantinib efficacy in a selected population of HCC patients with high METexpressing tumors identified using immunohistochemistry. However, several studies have questioned the mechanism of action of the drug, as they provided strong evidence that tivantinib acts on microtubule dynamics independently of MET and behaves as an antimitotic agent  (10) (11) (12) (13).
Drugs and cell viability assay Tivantinib, JNJ-38877605, PHA-665752, vinblastine and paclitaxel were purchased from Selleck Chemicals and dissolved in DMSO at 10 mM concentration. Cells were seeded in 96well plates at an optimal density (1500 to 3000 cells/well) to ensure that they were in exponential growth phase at the end of the experiment. After overnight incubation, cells were treated with 5 concentrations of each compound (10-fold dilution from 0. 001 to 10 µM in duplicates) using the HP D300 digital dispenser (Tecan). Cell viability was measured 48 hours after drug treatment by colorimetric MTS assay following the supplier's recommendations (Promega). Each experiment was repeated at least two times for each cell line and results were normalized on untreated cells. Curve fitting of dose-response data was performed using GraphPad Prism 6 Software and the two following classical parameters representative of drug sensitivity were derived: 1) the GI50 corresponding to the concentration of drug that inhibits 50% of cell viability and 2) the AUC corresponding to the area under the dose-response curve that provides an overall measure of cumulative response.
Western-blot analysis Cell protein extracts were prepared using RIPA lysis buffer containing protease and phosphatase inhibitors and quantified using the BCA Protein Assay kit (Pierce). Western-blot analyses were conducted using the following primary antibodies: MET (#8198), phospho-MET (Tyr1234/1235) (#3129), ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204) (#9101), AKT (#9272), phospho-AKT (Ser473) (#9271) and β-actin (#4967) was used as loading control. Protein of interest were detected using an anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (#7074) and the ECL Chemiluminescence Western Blotting Detection Kit (GE Healthcare), according to the provided protocol. Signal detection was performed using the ChemiDoc XRS system and the Image Lab software (BioRad). All antibodies were purchased from Cell Signaling Technology and used at 1:1000 dilution except secondary antibody which was used at 1:2000.
Reverse-phase protein array RPPA technology was used to quantify MET, phospho-MET Tyr1234-1235 and Ki67 protein level in the 35 liver cancer cell lines and 202 resected HCC as previously described  (16). Briefly, equal amounts of protein lysates were printed onto nitrocellulose covered slides. Five serial dilutions and two technical replicates per dilution were deposited for each sample. Arrays were revealed with anti-MET (Sc-10), anti-phospho-MET Tyr1234-1235 (CST3129) and anti-Ki67 (Dako M7240) antibodies. Quantification and normalization of RPPA data were performed using the NormaCurve method  (16).
Quantitative RT-PCR We analyzed total mRNA from 35 liver cell lines, 310 resected and advanced HCC and we assessed quality as previously described  (17). mRNA levels were determined by analyzing 500 ng of total RNA reverse transcribed using the High Capacity Transcription kit (Life technologies) and TaqMan predesigned assays (Life technologies), on Fluidigm 96. 96 (San Francisco) dynamic arrays using the BioMark Real-Time PCR system. A panel of 188 genes was analysed (Supplementary Table  3 ). Expression data (Ct values) were calculated using the Fluidigm Real-Time PCR Analysis software (4. 1. 3). Gene expression data was expressed with the 2 -ΔΔCT method relative to Ribosomal 18S (R18S) and the mean expression level of the corresponding gene in normal liver samples.
ACKNOWLEDGMENTS We thank surgeons and pathologists at  Henri Mondor hospital (APHP, Créteil, France ):  Julien Calderaro ,  Alexis Laurent ,  Daniel Azoulay ,  Alain Luciani ;  Jean-Frédéric Blanc ,  Charles Balabaud ,  Brigitte Le Bail ,  Christophe Laurent ,  Jean Saric ,  Nora Frulio ,  Claire Castaing  at  Bordeaux Hospital ; the  "Réseau Français des biobanques des tumeurs hépatiques" Bruno Clément ,  Valérie Paradis ,  Thomas Decaens ,  Simone Mathoulin-Pelissier ; the  Cancer Biobank of CHU Bordeaux , no. BRIF:  BB-0033-00036  and  CHU Henri Mondor  for contributing to the tissue collection. We thank  Floriane Bard ,  Caroline Lecerf ,  Bérengère Ouine  and  Audrey Criqui  for performing the RPPA experiments. We warmly thank  Frédéric Soysouvanh  for help in drug screening.
This work was supported by  Inserm , the  Ligue Nationale Contre le Cancer  ('' Equipe labellisée'' program ), the  Fondation Bettencourt-Schueller  ( Coup d'élan Award ),  Labex OncoImmunology (Investissement d'Avenir)  and  MUTHEC INCA translationnel. Samia Rekik  was supported by a fellowship from  ARC.
To this aim, the perceptual centers (i. e. , p-centers) of accented syllables were estimated using an automated procedure proposed by  Cummins and Port (1998). A p-center is defined as the time at which the occurrence of a syllable is perceived, which is most often around the vowel onset  (Morton et al. , 1976). It can be roughly estimated as the point corresponding to half of the amplitude rise before reaching maximum amplitude for a nuclear vowel  (Cummins and Port, 1998). The intervals between the estimated p-centers of accented syllables were calculated, and, if deviating from the 600 ms interval, they were manually adjusted by slightly shortening or lengthening silences or segmental material in the interval, using Praat software  (Boersma, 2001). A trial was created in which a stimulus was repeated with a 2-s pause between presentations.