dataesr/scientific-paragraphs-categorization huggingface dataset:
Influence of "primer sequences" on pyrosequencing results In this study, we included data from a previously unpublished study on the microbial composition of king penguins (M. L. Dewar et al. , unpubl. data)  and although the primer sequence used to amplify the V2-V3 region analysis of the sequence data produced has shown that the primer sets used in this study have a similar coverage of the bacterial taxa present in all penguin species as shown in Table  S3. Thus, we feel that the sequences used in the king penguin study do not influence the variation in microbial composition between king penguins and the other penguin species.
Acknowledgments The authors would like to thank  Stacey Adlard  and  Fabrice Le Bouard  from  BAS ;  Vincent Viblanc ,  Nelly Mallosse ,  Goetz Eichhorn , and  Ren e Groscolas  from  CNRS ; and  David Dewar ,  Kaye Dewar ,  Louise Kelly ,  Tiana Preston , and  Joel Maloney  for assistance with sample collection of fecal samples from  Bird Island ,  Possession Island , and  Phillip Island. The authors also thank  Holsworth Wildlife Research Endowment, Snowtown Valley Ltd. Pty, Centre for Molecular and Medical Research (Deakin University ), and the  French Polar Institute (IPEV)  (project No. 119 ) for financial and logistic support. Ethics for collection of samples was obtained from  Terres Australes et Antarctiques Franc ßaises ,  BAS/University of Cambridge Ethics Committee, and Deakin Universities Animal Ethics Committees.
S100A9 tetramer stimulation potentiates anti-HIV-1mediated NK cell activity We have previously shown that the engagement of the NK CD85j receptor with non-HLA class-I ligand(s) plays a critical role in the anti-HIV-1-mediated NK cell activity on MDDC  [20]. Since S100A9 proteins modulate NK cell activity (Figure  4 ), we then tested the capacity of S100A9 stimulated NK cells to control viral replication (Figure  5 ). Viral replication, measured by GFP expression, was decreased in MDDC after 7 days of culture with NK cells compared to MDDC cultured without NK cells (12. 5% vs 27. 9% HIV/GFP + MDDC, respectively, data not shown). However, S100A9 monomer-or tetramer-stimulated NK cells do not have a better capacity to control HIV-1 replication in MDDC than unstimulated NK cells (Figure  5A ) instead, S100A9 monomer stimulated-NK cells seem to slightly increase HIV-1 viral replication.
Chip-captured proteins were air-dried and covered with a matrix (3,5-dimethoxy-4-hydroxycinnapynic acid in 50% acetonitrile and 0. 5% trifluoroacetic acid), used as an absorbent for laser energy. The ionized and desorbed proteins were detected and their molecular masses displayed on the proteogram as peaks. SELDI-TOF-MS analysis was done with the Protein-Chip Biology System II software (PBS II) and Ciphergen Peaks software.
Acknowledgments We thank all the donors for their participation in this study. We thank  Dr. O. Schwartz  for providing some HIV-1 molecular clones,  P. Lenormand  for protein sequencing and  J. d'Alayer  for protein chip assays. We thank  Dr. G. Pancino ,  Dr. E. Menu  and  Dr. M. Müller-Trutwin  for their comments on the manuscript. This work was supported by  Agence Nationale de Recherche contre le SIDA et les hépatites virales (ANRS).
The genotype data of 7054 single nucleotide polymorphism (SNP) loci in 40 tomato lines, including inbred lines, F 1 hybrids, and wild relatives, were collected using Illumina's Infinium and GoldenGate assay platforms, the latter of which was utilized in our previous study. The dendrogram based on the genotype data corresponded well to the breeding types of tomato and wild relatives. The SNPs were classified into six categories according to their positions in the genes predicted on the tomato genome sequence. The genes with SNPs were annotated by homology searches against the nucleotide and protein databases, as well as by domain searches, and they were classified into the functional categories defined by the NCBI's eukaryotic orthologous groups (KOG). To infer the SNPs' effects on the gene functions, the three-dimensional structures of the 843 proteins that were encoded by the genes with SNPs causing missense mutations were constructed by homology modelling, and 200 of these proteins were considered to carry non-synonymous amino acid substitutions in the predicted functional sites.
SNPs in upstream and downstream regions might cause phenotypic variations because they often activate or suppress gene expression by causing substitutions in regulatory sequences, e. g. promoters and terminators. Therefore, investigations of the SNPs related to functional changes of genes are considered essential for clarifying the functional genomics of organisms, including plants. In human genetics, the categorized SNPs associated with human diseases have been released from the F-SNP database (http://compbio. cs. queensu. ca/F-SNP/). 4 ased on the support vector machine models, the sequence profiles and the structural stability of proteins whose functions are changed by non-synonymous substitutions have also been determined and are reported on the SNP3D website (http://www. snps3d. org).
SNP genotyping Genome-wide SNP genotyping was performed using the Infinium assay (Illumina Inc. ) that was developed by the Solanaceae Coordinated Agricultural Project (SolCAP),  8  according to the manufacturer's standard protocol. The probe sequences and SNP information are available from SolCAP (http://solcap. msu. edu) and in Supplementary Table  S1. The data obtained from the assays were analysed using the Genotyping Module of the GenomeStudio software (Illumina Inc. ).
Genetic diversity analysis and detection of linkage disequilibrium (LD) The Hamming distances  13  between each pair of the 40 tomato lines tested were calculated to generate the distance matrix based on the genotyping data obtained in this study. A neighbour-joining tree was generated using MEGA 5 software. 14 The heterozygosity (HZ) values were calculated by the equation HZ i ¼ 1 À X i j¼1 p 2 ij ; where p ij is the frequency of the jth of the i allele. Linkage disequilibrium (LD) in the tomato lines was detected by the r 2 and D 0 values calculated using the Tassel program. 15.
Inference of SNP effects on protein and gene functions The amino acid sequences of the genes were searched against the Protein Data Bank (PDB) (http:// www. rcsb. org/pdb/home/home. do) by Blastp  16  with an E-value cutoff of 1E-10. The amino acid sequences were further aligned on those of the homologous proteins by Fasta36 program,  17  and homology modelling was performed by Modeller 9. 10 software  18  based on the alignment with default parameters. The functional sites on the constructed protein structures were predicted using FPocket software  19  with default parameters. The functions of proteins were predicted by combining the results obtained by the Blastp searches with an E-value cutoff of 1E-10 against the databases NR (non-redundant amino acid sequence database released by NCBI) (http://www.
SNP genotyping by high-throughput assay technologies Of the 7720 scorable SNPs defined by the Infinium assay, 7617 (98. 7%) were successfully genotyped, and 6374 (82. 6%) showed polymorphism among the 40 tested lines. In our previous study using the GoldenGate assay, 916 of 1338 SNP loci (68. 5%) showed polymorphism among the 27 tomato lines. 12 mong the 916 SNP loci on the GoldenGate assay, 236 were duplicated on the Infinium assay; therefore, the genotype data of the specific 680 SNP loci on the GoldenGate assay were analysed with the 6374 SNPs of the Infinium assay. In total, polymorphic data were successfully obtained at 7054 SNP loci. The numbers of SNPs identified between each pair of the 40 lines are shown in Supplementary Table  S2. The average number of polymorphic loci between each pair of the 40 lines was 4270, whereas that between each pair of the 30 cultivated tomatoes, the 2 lines of S.
Inference of direct substitution effects on protein functions The amino acid residues directly altering protein functions were inferred according to the locations of substitutions on the functional sites of protein structures. A total of 1271 cSNP loci that altered the charges or polarities on amino acid residues were identified in 1108 genes. Of these 1108 genes, the three-dimensional structures of 843 corresponding proteins were constructed by homology modelling using Modeller v9. 10 software. 18 Furthermore, the 230 Genome-Wide SNP Functional Analysis in Tomato [Vol. 20, candidate functional sites were predicted by FPocket software. 19 hese analyses together revealed 215 cSNP loci that were candidate functional sites on 200 proteins (Supplementary Table  S1 ). The genes whose cSNPs were located on candidate functional sites have the potential to be applied as functional markers because they could directly affect the protein functions.
Therefore, differences in the cSNP distribution on the 25 KOG categories were investigated according to the cSNP positions on functional or other sites (Fig. 6 ). Significantly different distributions were observed on KOG O ( posttranslational modification, protein turnover, and chaperones), KOG C (energy production and conversion), KOG G (carbohydrate transport and metabolism), KOG E (amino acid transport and metabolism), and KOG Q (secondary metabolites biosynthesis, transport, and catabolism). Solyc09g010080. 2. 1, also known as brix9-2-5, 29,30 controls fruit sugar content and is involved in KOG G (carbohydrate transport and metabolism). It is also annotated as beta-fructofuranosidase. The three-dimensional structure of Solyc09g010080 was built by homology modelling based on the crystal structure of a cell-wall invertase from Arabidopsis thaliana (PDB id: 2ac1) (Fig.
7 ). The identity of the amino acid sequences between Solyc09g010080 and 2ac1 was 51. 0% in 92. 6% length coverage. In the constructed structure, 28 pockets were found as candidate functional sites by the FPocket program. 19 This program usually detects several candidates for functional sites, which are called pockets, in a protein structure because there are many small or large clefts on the surface of proteins. In a previous study,  29, 30  three amino acid substitutions (Gln297, Asn315, and Leu322) caused by cSNPs were found in brix9-2-5 between S. lycopersicum and S. pennellii. Asn315, which is one of the three amino acid residues, was identified on pocket 8 that was a relatively larger pocket that included 15 amino acid residues (Asn315, Leu316, Ser317, Lys319, Ser430, Tyr431, Lys432, Ile433, Val459, Asp460, Val461, Asp462, Leu463, Asp465, and Lys488).
The amino acid residue at the 315th position was altered from Asn to Asp, and the amino acid property was changed from 'polar' to 'negatively charged (acidic). ' The amino acid residue at the 315th position was Asp in the wild tomatoes and S. lycopersicum var cerasiforme (WIR25/30 and TOMJPF00009), whereas it was Asn in the other tomatoes. Aspartic acid residues play important roles in the catalytic sites of proteins, and, therefore, the activities of Solyc09g010080 might differ between the wild and cultivated tomatoes. In this study, we predicted the functions of 7054 SNPs identified by array assays. Although this was the total number of SNPs we investigated, we expect that greater numbers of SNPs exist on the tomato genome. The advance of NGS technologies has enabled the discovery of genome-wide SNPs by the re-sequencing of multiple tomato lines.
Acknowledgements: We are grateful to  Dr Ariizumi  ( National BioResource Project, Japan ),  Dr Saito  ( NARO Institute of Vegetable and Tea Sciences, Japan ),  Dr Tam  ( Tomato Genetic Resource Center, USA ), and  Dr Tanksley  ( Cornell University, USA ) for providing plant materials. The  SNP BeadChip was developed by Illumina 's technology and the  USDA-NIFA Agriculture and Food Research Initiative (AFRI)  sponsored Solanaceae Coordinated Agricultural Project. Supplementary data: Supplementary Data are available at www. dnaresearch. oxfordjournals. org.
Sequence processing, assembly and virus genome identification Sequence read processing, assembly and virus genome identification were conducted using a custom bioinformatics pipeline. Trimmed sRNA reads were assembled using Velvet  [16]  and contigs were compared to a database comprising all National Center for Biotechnology Information (NCBI) RNA virus proteins using blastx  [17]. For the total RNA samples, raw Illumina reads were trimmed using the FASTX-Toolkit (see hannonlab. cshl. edu/fastx_toolkit), first using fastq_quality_trimmer to trim sequences from the 3' end back to the first position with a quality score greater than or equal to 28, then using fastq_quality_filter to remove any reads with greater than 10% of positions having a quality score ≤ 20, and finally any reads still containing 'N' characters were removed.
Of the order of 150-200 million useable reads, with a mean trimmed length of 96 nt per read, were obtained per sample. Reads were then assembled using Velvet  [16]  with k-mer values ranging from 19 to 29. Contigs were compared to a database comprising all NCBI RNA virus RefSeq proteins using blastx  [17, 18]. Long contigs (> 600 nt) with, initially, blastx evalues < 0. 01 were extracted. Using blastx, these contigs were compared with a cellular proteome database (NCBI, nonredundant protein database), and contigs with better matches to cellular proteins than to RNA virus proteins were removed. The remaining contigs were inspected manually and selected contigs of interest were concatenated with other partially overlapping contigs where possible. These semi-manual assemblies were also compared with automated Oases  [19]  and PRICE  [20]  assemblies.
Assemblies were tested for possible further 5' and 3' extensions by iteratively searching for reads with 23+ nt perfect terminal overlaps. This enabled the joining of two contigs to form KF298274 (verified by similarity in both contigs to phlebovirus L proteins, and complementary 5' and 3' ends in the assembled KF298274, as expected for members of the family Bunyaviridae), and 5' and 3' extensions of KF298265 (verified by Sanger sequencing). 1  contigs were compared to the NCBI non-redundant nucleotide database, and all showed greatest similarity to RNA virus sequences with e-values < 10 -9. To test for (known) genome-integrated sequences, contigs were also compared to the NCBI non-redundant nucleotide (all taxa), transcriptome shotgun assembly (all taxa) and whole genome shotgun contigs (Diptera) databases using tblastn and blastn.
To test for chimeric sequences (e. g. mis-assembled or transcripts derived from genome-integrated sequences with flanking non-virally derived sequences), regions of contigs that were too divergent to blast-match to known viral sequences were separately compared to the above databases using tblastn and, where relevant, blastn. It should be noted that, due to incompleteness within the public databases, such methods cannot conclusively establish that sequences are not derived from genomeintegrated sequences. To test for DNA contamination in the non-DNase treated total RNA samples, reads from the small-RNA-enriched samples were mapped back to the assembled contigs; all 14 contigs were supported by between 19 (KF298270) and ~140,000 (KF298271) reads, and mapped reads were distributed throughout the length of contigs.
Validation and completion of viral genome sequences with Sanger sequencing To further test for chimeric sequences and other misassemblies, selected contigs (see Table  1 ) were validated via Sanger sequencing of overlapping RT-PCR products (Genbank accession numbers KF298278 to KF298284). Samples were DNase-treated prior to RT-PCR The PCR products were A-tailed and cloned into the TA vector -pGemTeasy (Promega, Madison U. S. A. ), followed by transformation into DH5α cells. Plasmid DNA were extracted by Plasmid DNA miniprep kit (NBS Biologicals Ltd. Cambridge U. K. ) and sequenced with both M13F and M13R primers as well as internal primers when necessary (primer sequences available from authors on request).
Phylogenetic and conservation analyses Sequences in GenBank related to AACV, CAZV and other selected contigs were identified using tblastn  [17, 18]. Multiple sequence alignment was performed via MUSCLE  [21]. Regions of ambiguous alignment were removed using G-Blocks  [22]. Maximum likelihood (ML) phylogenetic trees were estimated via the Bayesian Markov chain Monte Carlo (MCMC) method implemented in MrBayes. All parameters were estimated from the data under default priors and Markov chains were run for a minimum of 20 million generations, with the first 10% of samples discarded as burn-in. Support for nodes was assessed using posterior probability values calculated in MrBayes. All phylogenetic analyses were carried out on the freely available Bioportal server (www. bioportal. uio. no). Due to the highly divergent nature of many of the sequences, alignments were also conducted via COBALT, using conserved domain and local sequence similarity information  [23]  and neighbour-joining trees constructed via PAUP  [24]  for comparison.
As such, this likely represents a transcribed genome-integrated sequence. In silico, we were unable to link this sequence with KF298267, and were also unable to otherwise extend KF298267 further 5'-ward. Thus, KF298267 may also be derived from a genome-integrated sequence. Indeed, flavivirus-derived integrated sequences have been identified previously in insect genomes  [42, 43] , and a frameshift-interrupted flavivirus-like sequence fragment (KA183058;  [44] ) exists in a Chironomus riparius transcript shotgun assembly database. Although we identified 15 SNPs in KF298267 (10 synonymous and 5 nonsynonymous), this is not necessarily inconsistent with a genome-integrated sequence, as genome-integrated sequences of viral origin are sometimes subject to exaptation and purifying selection  [43, 45].
l. mosquitoes from a farm/stables site) (KF298266; 4373 nt), which may be suggestive of a bona fide replicating virus. Related sequence fragments are present in Anopheles funestus transcriptome shotgun assembly databases  [50] , notably the non-overlapping sequences EZ920122 (269 nt; 85% amino acid identity to KF298266) and EZ922704 (207 nt; 68% amino acid identity to KF298266). It is not clear whether such sequences derive from cellular transcripts or from infecting viruses. Although orbivirus segments have conserved (but not fully complementary) 5'and 3'-terminal sequences of around 6 nt, the motifs differ slightly between species, and considerably between different genera within the family Reoviridae. The 5' and 3' termini of KF298266 and KF298273 are not obviously similar to orbivirus conserved motifs, so it is not clear whether or not these sequences represent complete viral segments.
Blast analysis of KF298277 revealed related sequence fragments in AAGE02000678, a Stegomyia aegypti whole genome shotgun sequence. Here, two adjacent ORF2-related fragments of ~250 and ~210 codons are present in AAGE02000678, separated by an unrelated insert of ~5500 nt that includes sequences similar to retrotransposons. Thus (bar misassembly) genome integration of related viruses appears to occur. A number of related viral sequences were also identified in searches of the NCBI transcriptome shotgun assembly database, including JP306102 from Lepeophtheirus salmonis (salmon louse, an arthropod), JP089782 from Mengenilla moldrzyki (a strepsipteran insect), and EZ959541, HP663044, EZ941741, EZ958863 and HP643964 from Bemisia tabaci (silverleaf whitefly, an hemipteran insect). Potentially, these sequence fragments may derive from either transcribed genome-integrated sequences or from infecting viruses.
Contigs KF298275 (2507 nt) and KF298276 (3074 nt), which have about 40% amino acid identity, were each spanned (up to 835 and 1022 codons respectively) by an open reading frame, the translation of which bears similarity to the RdRp of Saccharomyces cerevisiae 20S and 23S RNA narnaviruses. The sequences are nonetheless highly divergent from Saccharomyces cerevisiae 20S and 23S RNA narnaviruses, with amino acid identities, averaged over the whole ORF, of just 12-15%. By searching the NCBI Transcriptome Shotgun Assembly (TSA) database, we identified six other sequences (lengths 2159 to 3097 nt) related to KF298275 and KF298276, namely GACI01002802 (from Uromyces appendiculatus), GACM01002912 (from Phakopsora pachyrhizi), and GAIR01011807, GAIR01012025, GAIR01012062 and GAIS01005902 (all from Puccinia striiformis).
They have bipartite dsRNA genomes with one segment encoding the RdRp and the other segment encoding a capsid protein  [56]. We detected partitivirus-like sequences in samples 7 and 9, one of which, KF298269 (1503 nt), is listed in Table  1. This sequence potentially represents a full-or near full-length RdRp-encoding segment. However, despite relatively high coverage (98-fold), no SNPs were detected for this contig. Blast analysis revealed many related sequences in the NCBI transcriptome shotgun assembly and whole genome shotgun sequence databases. Some of the former may derive from infecting viruses. Nonetheless, partitivirus sequences are commonly integrated into the genomes of cellular organisms including arthropods  [57]. KF298269 had notably higher similarity (87% coverage, 42% identity) to a region of at least one of the whole genome shotgun sequences (AAPT01020539; Drosophila grimshawi; see also 57) than to the most closely related virus sequence in GenBank (Table  1 ).
Tblastn against non-redundant (nr) database, all taxa. d. Predicted to be expressed as join(11 1618,1620. 3533) via +1 ribosomal frameshifting on UUU_C. e. Hit to AB018086 (Chuzan orbivirus segment 1) has a slightly lower e-value (6 x 10 -47) but greater coverage (83% coverage; 24% identity). f. Predicted to be expressed as join(92 1891,1891. 2694) via -1 ribosomal frameshifting on U_UUU_UUA.
Predicted to be expressed as a fusion with the ORF 742. 3378 via -1 ribosomal frameshifting on G_GAA_AAC. k. Related sequences are generally associated with mosquitoes of the genus Culex. l. Verified by Sanger sequencing -GenBank accessions KF298278 to KF298284. doi: 10. 1371/journal. pone. 0080720. t001.
SC was funded by a  Wellcome Trust   Sir Henry Wellcome Postdoctoral Fellowship  [082744]. See www. wellcome. ac. uk. Work in the AEF lab is funded by grants from the  Wellcome Trust  [ 088789 ] and the  U. K. Biotechnology and Biological Research Council (BBSRC)  [ BB/J007072/1  and  BB/ J015652/1 ]. See www. wellcome. ac. uk and www. bbscr. ac. uk respectively. BYWC is funded by an  EMBO   Long-Term Postdoctoral Fellowship and a Sir Henry Wellcome Postdoctoral Fellowship  [096082]. See www. embo. organd www. wellcome. ac. uk respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
c. Tblastn against non-redundant (nr) database, all taxa. d. Predicted to be expressed as join(11 1618,1620. 3533) via +1 ribosomal frameshifting on UUU_C. e. Hit to AB018086 (Chuzan orbivirus segment 1) has a slightly lower e-value (6 x 10 -47) but greater coverage (83% coverage; 24% identity). f. Predicted to be expressed as join(92 1891,1891. 2694) via -1 ribosomal frameshifting on U_UUU_UUA.
j. Predicted to be expressed as a fusion with the ORF 742. 3378 via -1 ribosomal frameshifting on G_GAA_AAC. k. Related sequences are generally associated with mosquitoes of the genus Culex. l. Verified by Sanger sequencing -GenBank accessions KF298278 to KF298284. doi: 10. 1371/journal. pone. 0080720. t001.
, Miami, FL, USA) with an MPL (Multi-Platform Loader) auto-sampler for a 96-well plate format. For quality control, verification of the flow cytometer's optical alignment and fluidics system using Flow-Check Fluorospheres (Beckman Coulter Inc. ) was run every time before cell samples. PI (DNA content) fluorescence were triggered by a 488 nm argon laser and detected and measured in the FL3 (670LP) channel. Readings from 10,000 cells were collected and analyzed by Quanta Analysis software (Beckman Coulter Inc. ). SubG0/G1 cell populations representing apoptotic cells were quantified.