dataesr/scientific-paragraphs-categorization huggingface dataset:
Identity of purified HILS1 protein was confirmed by Western blot analysis with anti-HILS1 antibodies (Fig. 1d ). To further confirm the identity of the purified protein, we performed MALDI-TOF MS analysis. Database search using MASCOT indicated 79% peptide coverage for rat HILS1 protein (Table  1 ). Since HILS1 is a newly reported linker histone variant whose structure and function are not known, we were curious to understand the secondary structure of HILS1   and compare it with well-characterized linker histones H1d and H1t. Multiple sequence alignment of rat H1d, H1t, and HILS1 proteins revealed significant divergence particularly in the N-and C-terminal domains. Rat HILS1 and H1t proteins showed amino acid sequence similarity of 30. 18%, while rat HILS1 and H1d exhibit 30. 77% sequence similarity (Fig.
Rat H1d and H1t proteins were purified using Ni-NTA agarose column followed by binding with heparin agarose and HILS1 protein was purified by one-step Ni-NTA affinity chromatography as described in methods. f Sequence alignment of rat linker histones H1d, H1t, and HILS1, aligned by Clustal Omega software. Globular domain has been highlighted in red. S/TPXK motif in the CTD has been highlighted in orange. Rat H1d possesses four S/TPXK motifs, rat H1t has two, whereas rat HILS1 lacks S/TPXK motif in the CTD. g-i, Circular dichroism spectra of rat recombinant linker histones H1d, H1t, and HILS1, respectively. A 200 µg/ml solution of purified histone H1 subtypes was used for recording the CD spectrum in a Jasco J-810 spectropolarimeter. The spectra were recorded in 10 mM sodium phosphate buffer, pH 7.
HILS1 shows unique association with LINE-1 repeats Since most of the HILS1 peaks were present in intergenic regions, we then aligned the HILS1 peaks to different repetitive DNA elements present in the UCSC repeat element database and annotated with the help of intersectBed (Bed tools) and HOMER v4. 7. We observed that most of the peaks were present in LINE-1 elements, followed by LTR and satellite repeats (Fig. 4k ; Additional file 7: Table  S6 ). We also looked for enrichment at 10 kb resolution for selected regions present on different chromosomes and belonging to different categories (Fig. 5a , Additional file 8: Figure  S3A  and Additional file 9: Figure  S3B ). Even though we observed some background signals in the input, HILS1 ChIP peaks showed more enrichment signals compared to the input control.
qPCR analysis showed the specific enrichment of both the modifications in HILS1-associated loci (genomic positions are same as detailed in Fig. 6 ) in separate IP experiments and the negative control corresponds to a region in chr 8 depleted of HILS1. Data were quantified as % of input. Results are from three independent experiments, and error bars represent standard deviation. Asterisk shows comparison with negative control IgG for single IP, and # shows comparison with IgG+antibody2 for reChIP. ***P ≤ 0. 001; **P ≤ 0. 01; *P ≤ 0. 05  [41, 52]. HILS1 was shown to bind to reconstituted mononucleosomes and also produces chromatosome stop during MNase digestion, although its binding to the mononucleosome appeared less efficient than somatic linker histone H1a  [41]. Previous study from our laboratory found that rat HILS1 exhibits extensive posttranslational modifications that include many phosphorylation and acetylation sites in all the three structural domains  [6].
Genomic organization of Hils1 and multiple sequence alignment Information on the genomic organization of the rat Hils1 gene (NCBI gene ID: 690026, mRNA: NM_001109565. 1) was obtained from the NCBI database. Domain organization of this protein was constructed based on the sequence comparison with chicken H5 (Uniprot Id:P02259)  [6]. Multiple sequence alignment was performed by Clustal Omega software using rat H1d (Uniprot Id: P15865), rat H1t (Uniprot Id: P06349), and rat HILS1(Uniprot Id: D3ZZW6) amino acid sequences.
Further, gel pieces were dehydrated with 50 μl of acetonitrile for 15 min and dried completely in the Speed-Vac. Sequencing grade trypsin (150 ng) was added to gel pieces and after digestion at 37 °C overnight, the resulting proteolytic peptides were subjected to hydrophobic extraction (50 μl of 50% ACN, 5% formic acid), and the extracted peptides were analyzed by mass spectrometry. To identify the protein, spectra were analyzed by MASCOT software using NCBInr database. The search parameters included up to four missed cleavage sites, variable oxidation states of methionine, and carbamidomethyl as fixed modification.
ChIP-seq data analysis ChIP and input DNA libraries were prepared using the ChIP-seq Sample Preparation Kit (Illumina). Briefly, 50 ng of ChIP and input DNA were repaired to overhang a 3′-dA and adapters were ligated to the end of DNA fragments. DNA fragments of 100-300 bp were size selected after PCR amplification. Library DNA was purified using Qiagen DNA purification kit. Sequencing was performed with Illumina HiSeq 1000 system using paired-end reads of 101nt length. Sequenced reads containing adaptor sequences were trimmed and filtered with Phred quality threshold as 20. Read pairs falling below 20 bp after clipping were removed. Clean reads were uniquely aligned against UCSC Rattus norvegicus genome (rn5, Mar. 2012) using Bowtie2 (version 2. 2. 3) with no mixed and no discordant parameters  [92, 93].
For finding the overlapping peaks, Bed tools Intersect was used with 100 bp between summits and only overlapping peaks  (32, 731)  were used for subsequent analysis. Chromosome-wise distribution plots were generated using custom R script. Genome browser view for chromosome-wise peak distribution was generated using UCSC genome browser. HILS1 peak distribution was identified at genomic locations enriched in CpG islands, repetitive elements, exons, introns, intergenic regions, 3′UTR and 5′UTR using HOMER v4. 7 (for annotation) and Bed tools (for identifying the intersections). Box plots were created using R 3. 3. 2 statistical software. De novo motif identification was done using 300 bp regions surrounding overlapping peak summits as target set and shuffled set of same overlap sequences as background set with MEME software  [95].
The PCR conditions were as follows: 95 °C for 4 min; 95 °C for 30 s, appropriate annealing temperature for 30 s and 72 °C for 30 s, for 35 cycles in a total volume of 10 µl. The C t value (number of cycles required for the fluorescent signal to cross the threshold) was recorded in the experimental report after analysis by Rotor-Gene 6000 software. The C t values of the duplicates showed minimal variability, and C t values were used for calculating the percent of input using the following formula. In sequential ChIP experiments, the amount of genomic DNA co-precipitated with specific antibody was calculated in comparison with the corresponding input DNA used for each immunoprecipitation using the same formula. Primers used for sequential ChIP-qPCR are same as listed in Additional file 13: Table  S9.
RT-PCR virus detection Total RNAs were used for the detection of the aureusvirus and for the amplification of targeted viral genome parts using RT-PCR. Complementary DNAs were synthetized using random hexamers and the BioScript reverse transcriptase (Bioline). PCR amplification involved the ElAur1F/ElAur1R primer pair targeting the RdRp gene for virus detection (expected PCR fragment of 457 bp; nt positions 716-1172 of the ElAV1-B15 genome, MG967280); the ElAur1-RdRpF/ElAur1-RdRpR primer pair targeting the RdRp domain (1215 bp; nt positions 782-1996); or the ElAur1-CPF/ElAur1-CPR pair targeting the full CP gene (1333 bp, nt positions 2285-3617) (for list of primers see Table  1 ). These primers were designed based on the NGS data using Primer-BLAST  [30]. All PCR amplifications were performed using the MyTaq DNA polymerase (Bioline) and 0.
Bioinformatic analysis of sequence data De novo assembly of high quality reads was performed with the CLC Genomics Workbench 9, using default parameters and a minimum contig length of 200 bp. The obtained contigs were annotated by BLASTN and BLASTX analyses  [35]  against the GenBank nt and nr databases  [36]. Sanger sequencing reads were assembled using the Seqman Pro Assembler, Lasergene v. 12. 3. 1. 4 (DNASTAR, Inc. ). Multiple alignments, pairwise genetic distance computations, model selections, all phylogenetic analyses and tree visualisations were performed using Mega v. 7. 0. 14 [37]. The obtained full genomic sequence and/or partial sequences were aligned with all available GenBank aureusvirus sequences and with reference Tombusviridae sequences using the ClustalW algorithm  [38]. Phylogenetic analyses were performed using the neighbor-joining method  [39, 40]  and the Tamura 3-parameter model  [41]  for nucleotide (nt) sequences or the JTT model  [42]  for amino acid (aa) sequences with 500 bootstrap replications.
Results During the survey for the viruses infecting elderberry trees in the Czech Republic, a tree (marked as B15) showing virus-like symptoms such as leaf chlorosis, leaf dwarfing and reduced, non-uniform fruit development, was selected for detailed NGS analysis. Highly purified double-stranded RNAs were sequenced using the Illumina HiSeq2500 platform. In total, 27,747,697 reads (average length 100 nt) were obtained and were assembled in 4638 contigs, two of which showed high identity values with aureusvirus sequences available in GenBank  A BLAST analysis of the nucleotide sequence (nt) of the RdRp-encoding region showed highest identity (70. 6%) with the aureusviruses Pothos latent virus isolate pigeonpea (AJ243370) and Yam spherical virus (NC_022895). Comparisons of the deduced amino acid sequence showed respectively 75.
database. Contig B15-325, 2137 nt in length (1346 reads, average coverage 60x) showed 78. 2% identity with the RNA-dependent RNA polymerase gene of Pothos latent virus (PoLV, isolate lisianthus, AB602348); contig B15-40, 2436 nt in length (1801 reads, 73x coverage) showed 83. 2% identity with the partial coat protein sequence of PoLV-pigeonpea (AJ243370). The scaffolding of the overlapping ends these contigs (Fig 1), together with 5´-RACE and 3´-RACE PCR amplifications allowed us to obtain the complete genome sequence of the virus infecting the original B15 elderberry tree. The B15 isolate genome is 4512 nt long (3144 reads, average coverage 69x) and comprises five ORFs with the typical organisation of aureusviruses in the Tombusviridae family (Fig 1). The first open reading frame is 729 nt long (genome positions 103-831) and encodes a replication-associated protein of 242 aa (26.
This work was funded by  Ministry of Education, Youth and Sport (MEYS) ,  Czech Republic , grant number  LD15048 -Application of next generation sequencing in study of fruit tree viromes , and by  European Cooperation in Science and Technology (COST)   Action FA1407 -Application of next generation sequencing for the study and diagnosis of plant viral diseases in agriculture (DIVAS) ,  Short Term Scientific Mission (STSM)  Grant, both obtained by  Dana S ˇafařova. The work was also supported by the  Internal Grant Agency (IGA) of Palacky ´University, Olomouc , grant number  PRF-2018-005. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Angiostrongylus cantonensis, the rat lungworm, is a zoonotic pathogen that is one of the leading causes of eosinophilic meningitis worldwide. This parasite is regarded as an emerging pathogen with a global range expansion out of southeastern Asia post-WWII. To date, molecular systematic/phylogeographic studies on A. cantonensis have mainly used two mitochondrial (mtDNA) markers, cytochrome c oxidase 1 (CO1) and cytochrome b (CYTB), where the focus has largely been descriptive in terms of reporting local patterns of haplotype variants. In order to look for more global evolutionary patterns, we herein provide a collective phylogenetic assessment using the six available whole mtDNA genome samples that have been tagged as A. cantonensis, A. malaysiensis, or A. mackerrasae along with all other GenBank CO1 and CYTB partial sequences that carry these species identifiers.
In order to look for more global evolutionary patterns, we herein provide a collective phylogenetic assessment using the six available whole mtDNA genome samples that have been tagged as A. cantonensis, A. malaysiensis, or A. mackerrasae along with all other GenBank CO1 and CYTB partial sequences that carry these species identifiers. Our study focuses on these three species due to their morphological and life-history similarities  (Spratt, 2015 ; details given in the Discussion). Our results reveal three important complications that researchers will need to be aware of, or will need to resolve, prior to conducting future molecular evolutionary studies on A. cantonensis. These three problems are (i) incongruence between taxonomic identifications and mtDNA variants (haplotypes or whole mtDNA genome samples), (ii) the presence of a CYTB mtDNA pseudogene, and (iii) the need to verify A.
| Assembly of CYTB and CO1 datasets Available in GenBank, there are five mitochondrial genomes identified as either A. cantonensis (NC_013065 = GQ398121,  Lv et al. , 2012; AP017672, BioProject: PRJEB493; KT186242, Yong, Song, et al. , 2015; KT947978, Yong et al. , 2016)  or A. malaysiensis (KT947979 = NC_030332;  Yong et al. , 2016). A whole mtDNA genome identified as A. mackerrasae was discussed in  Aghazadeh et al. (2015) , but where the actual sequence was obtained from the appendix of the thesis by  Aghazadeh (2015). We refer to these six samples as the mtDNA genome reference samples. Sample information, for example, geographic location, and host species, of the six reference samples was obtained from the original publications or available information on GenBank and is included in Tables  S1  and S2.
As prior studies on A. cantonensis do not include both CO1 and CYTB, a primary goal was to determine the correspondence between CO1 and CYTB clades that have been identified in prior work. Because the mtDNA does not generally recombine  (Ballard & Whitlock, 2004) , we used the CYTB and CO1 sequences from the above six mtDNA genome reference samples to anchor CYTB or CO1 haplotypes obtained from previous studies. From GenBank, we assembled all available CYTB or CO1 sequences that have been tagged as A. cantonensis, A. malaysiensis, or A. mackerrasae and that have associated publications. Tables  S1  and S2  also contain the sample information for all these additional sequences obtained from GenBank. For outgroups, we used CYTB and CO1 sequences from the mtDNA genome samples of A. costaricensis (NC013067, AP017675, and KR827449) and A.
vasorum (NC_018602). Alignments were conducted with ClustalW in BioEdit  (Hall, 1999)  along with manual corrections made by eye. In aligning sequences, a haplotype from a particular study may have been subsumed (i. e. , there was an identical match in the overlapping region) by a longer sequence from another study. In such cases, we only used the longer sequence in the phylogenetic analyses. The matching GenBank haplotypes are indicated in Tables  S1  and S2. The CYTB alignment consisted of approximately 852 nucleotides. Primers that amplify this region of CYTB are described in  Dusitsittipon et al. (2017)  (also, see below). Due to the presence of indels in a CYTB pseudogene (see Results), the CYTB alignment was based on amino acid translation (translation table 5, the invertebrate mtDNA code, on GenBank) of the mtDNA sequences.
Subsequent nucleotide alignments (after back translation) were then kept in codon reading frame to facilitate downstream molecular evolution analyses. The CYTB dataset contained 38 unique in-group haplotypes (including pseudogene haplotypes; see Results) and three outgroup sequences. Table  S3  contains the CYTB alignment in fasta format. Because many of the available CO1 sequences were short (e. g. , ~300 bases), information with regard to the relationships among the six mtDNA genome reference samples would be lost if we trimmed the alignment to match the shortest sequence. As our primary interest was in assessing CO1 haplotype relationships with respect to the six mtDNA genome reference samples, the CO1 alignment retained the full-length sequences of reference samples. The resulting gapped sites (i.
e. , sites that flank the overlapping region) in such an alignment are treated as missing data in a maximum-likelihood phylogenetic analysis. We note that there were two CO1 sequences (MF000735 and MF000736) from  Dalton et al. (2017)  that did not overlap with the region of CO1 that has predominately been used in studies of A. cantonensis. As a result, the phylogenetic positions of the CO1 haplotypes of  Dalton et al. (2017)  should only be interpreted with respect to the six reference sequences and not the remaining CO1 haplotypes. The topology of the CO1 tree remains the same even if the sequences of  Dalton et al. (2017)  are excluded (results not shown). The CO1 dataset contained 41 unique in-group haplotypes and four outgroup sequences. Table  S4  contains the CO1 alignment in fasta format.
| Phylogenetic analyses Phylogenetic analyses were conducted in MEGA6 software  (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013)  after using the maximumlikelihood model selection (with the "use all sites' option") to determine the evolutionary substitution model. To confirm that both the CO1 and CYTB haplotypes from the six reference samples provided the same phylogenetic signal, we first conducted maximumlikelihood analyses using the TN93 + G and HKY + G models, respectively. For heuristic purposes and to determine whether the two mtDNA markers provided similar information, we calculated pairwise p-distance between the six reference samples and tested for a correlation between the CO1 and CYTB pairwise p-distances using a Mantel test. For the complete CO1 and CYTB datasets, maximum-likelihood analyses with the GTR + G and HKY + G models, respectively, were used for phylogenetic reconstruction.
One thousand bootstraps were used to assess nodal support. In our prior study  (Dusitsittipon et al. , 2017) , we found strong associations between nine nuclear microsatellite clusters and CYTB mtDNA clades. We follow the naming conventions of the clusters and clades identified in  Dusitsittipon et al. (2017)  to facilitate discussion in linking to CO1 clades. In general, we linked CO1 and CYTB clades based on the phylogenetic positions of the reference samples. However, bootstrap support was expected to be low in the complete CO1 dataset because of the presence of many short sequences (e. g. , ~300 bases). As a result, it is important to recognize that the positions of the short CO1 haplotypes are subject to change pending acquisition of new sequence data. Also, for the latter reason, we used a conservative approach where a link in the CO1 phylogeny was made only back to the node that contained the most recent common ancestor of a reference sequence unless a clade had >90% bootstrap support.
| Phylogenetic analyses CO1 and CYTB phylogenetic trees based on the six mtDNA genome reference samples were identical in topology (data not shown) and matched their relationships observed in the results based on the complete CO1 and CYTB datasets (Figures  1  and 2 ). There is a significant and very high correlation in CO1 and CYTB pairwise p-distances of the six reference samples (r =. 99; p <. 001; Table  2 ). Thus, as would be expected if there was no recombination, the two mtDNA genes show the same phylogenetic signal and are appropriate to use as anchors for the CYTB and CO1 haplotypes observed in previous studies. At the broadest level, two divergent clades are observed in the both the CO1 and CYTB datasets (Figures  1  and 2 ). Clade I is anchored by three reference mtDNA samples originally identified as A.
cantonensis (NC_013065, AP017672, and KT947978) and the sample identified as A. mackerrasae, which is very closely related to AP017672. Clade II is anchored by KT947979 (originally identified as A. malaysiensis;  Yong et al. , 2016)  and KT186242 (originally identified as A. cantonensis,  Yong, Song, et al. , 2015). A third clade, Clade III, was also identified in the CYTB dataset. Evidence is provided below that shows Clade III represents a CYTB pseudogene lineage within specimens that fall in Clade II. Comparing the CO1 and CYTB phylogenetic trees, the reference samples enabled the linking of four subclades and relative phylogenetic positions provided hypothesized links between two additional subclades. Within Clade I, Subclade I. 1 is anchored by the reference KT947978 (=CYTB Haplotype 6 of  Dusitsittipon et al.
We also highlight that the Cytb-F and Cytb-R primers were used in a study by  Yong, Eamsobhana, Song, et al. (2015)  for samples they identified as A. cantonensis and A. malaysiensis. However, upon downloading from GenBank, it was observed that all of  Yong, Eamsobhana, Song, et al. (2015)  sequences labeled as A. malaysiensis were marked as "unverified" on GenBank (Table  S1 ). Indeed, straight translation (i. e. , no gaps in the alignment are inserted) of these sequences revealed premature stop codons. One haplotype we observed, H24, was the same as their AMM_1TAK sample (GenBank # KP732098) (Figure  1 , Table  S1 ). Aligning these "unverified" sequences with our own samples showed eight different untranslatable haplotypes. After alignment of these eight to the reference mtDNA samples, the untranslatable haplotypes had four common regions with deletions of six, one, one, and two nucleotides in length.
With the exception of the A. mackerrasae reference (discussed below), all haplotypes (CYTB or CO1) that fall in Clade I originated from samples originally identified as A. cantonensis (Tables  S1  and S2 ). In contrast, samples that fall in Clade II were identified as either A. cantonensis or A. malaysiensis. In the CO1 phylogeny, several haplotypes from wild-collected larval samples that were identified as A. cantonensis in  Vitta et al. (2016)  fall into Clade II (Table  S2 , Figure  2 ). Vitta et al. (2016)   GenBank accession numbers are MG516589-MG516592 for H22-H25, respectively. Sample CMI 4 had H23 (Table  S4 ), but we did not obtain the corresponding CYTB haplotype from this sample. from  Rodpai et al. (2016) , also from wild-caught larval samples, fell into Clade II. Rodpai et al.
(2016)  referred to their samples as A. malaysiensis in conjunction with the finding of two diverged CO1 clades that corresponded to two 18S ribosomal DNA (rDNA) clades that had prior species-designated reference sequences (see below). Eamsobhana, Yong, et al. (2017)  identified adult, wild-caught samples as A. malaysiensis, and their samples also fall in Clade II (Table  S2 ; Figure  2 ). Notably, we highlight that the reference sequence KT186242  (Yong, Song, et al. , 2015)  was based on the whole-genome sequence of a Thai laboratory isolate identified as A. cantonensis in Yong,  Eamsobhana, Lim, et al. (2015). The same group in the subsequent year  (Yong et al. , 2016)    F I G U R E 1 Maximum-likelihood phylogenetic tree of the complete mtDNA CYTB dataset (Tables  S1  and S3 ).
Yong, Eamsobhana, Song, et al. (2015)  used the Cytb-F and Cytb-R primers on samples identified as A. malaysiensis and recovered untranslatable haplotypes. As shown in our analyses, these haplotypes of  Yong, Eamsobhana, Song, et al. (2015)  fall into Clade III, that is, the pseudogene clade. Fortunately, F I G U R E 2 Maximum-likelihood phylogenetic tree of the complete mtDNA CO1 dataset (Tables  S2  and S4 ). The six reference mtDNA samples are indicated by circles. Clade I is anchored by three reference mtDNA samples originally identified as Angiostrongylus cantonensis (NC_013065, AP017672, and KT947978) and the sample identified as A. mackerrasae. Clade I is referred to as A. cantonensis (see main text for justification). Clade II is anchored by KT947979 (originally identified as A. malaysiensis;  Yong et al.