dataesr/scientific-paragraphs-categorization huggingface dataset:
BACKGROUND Biodiversity screens and phylogenetic studies are dependent on reliable DNA sequences in public databases. Biological collections possess vouchered specimens with a traceable history. Therefore, DNA sequencing of samples available at institutional collections can greatly contribute to taxonomy, and studies on evolution and biodiversity. METHODS We sequenced part of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and the SSU rRNA (V7/ V8) genes from 102 trypanosomatid cultures, which are available on request at www. colprot. fiocruz. br.
(3, 8, 13) t is clear nowadays that trypanosomatid taxonomy, previously strongly based on morphotypes, host specificity or site/mode of host colonisation, must also be supported by phylogenetic inferences. To this end, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and the V7/V8 variable region of the 18S small subunit ribosomal RNA (SSU) have been widely used and have been advocated as barcodes for trypanosomatid taxonomy, while the spliced leader (SL) RNA and the internal transcribed spacer (ITS) region of the ribosomal RNA are suitable to analyse strain or isolate diversity. (3, 8, 14) The use of these markers allows the identification of species through comparison with sequences available in public databases. However, it is not uncommon that the public databases lack one or more of the aforementioned molecular markers, or even worse, the associated data of the sequenced organism is imprecise, incomplete, outdated or even incorrect.
The PCRs were subjected to reagents and temperature adjustments to amplify the region of each of the molecular markers used for each species analysed. The amplified DNA segments were visualised by staining with GelRed after electrophoresis separation in 1. 5% agarose gel in a horizontal vessel, prepared at TBE solution (1. 1 M Tris, 0. 9 M Borate and 25 mM ethylenediamine tetra-acetic acid, pH 8. 3) for 50 min at 90 V. PCR reaction products were purified by Wizard SV Gel and PCR Clean-Up System kit, and sequenced in both directions using the BigDye Terminator v3. 1 cycle sequencing kit and loaded in the ABI 3730 Sequencing Platform at Fundação Oswaldo Cruz. Sequences were analysed and edited using the SeqMan software (DNASTAR software package, DNASTAR Inc. , Madison, WI, USA).
Molecular identification and phylogenetic analysis -The generated sequences were compared with available trypanosomatid sequences in Genbank by using BLASTn search. For phylogenetic analyses using maximum likelihood (ML) or Bayesian (BI) inferences, the DNA sequences obtained in this study [Supplementary data (Table  I )] or retrieved from Genbank [Supplementary data (Table  II )] were aligned using multiple sequence alignment with high accuracy and high throughput (MAFFT) online server. (16) The alignment was automatically trimmed using METAPIGA v. 3. 1. (17) hree alignments were created: (i) V7/V8SSU rRNA sequences, (ii) gGAPDH sequences and (iii) concatenated sequences. The final dataset, which compiles sequences generated in this study and sequences retrieved from GenBank, contained 134 specimens and 410 nucleotide positions (V7/V8), 159 specimens and 720 nucleotide positions (gGAPDH) and 161 taxa and 1130 nucleotides (concatenated analysis).
The evolutionary model (GTR+I+G -General Time Reversible with Invariant and Gamma distribution) was selected using Akaike criterion in METAPIGA v2. 0. ML phylogenetic inference was performed in METAPIGA v2. 0 with the selected model and 1,000 bootstrap replicates. Other parameters were used as default states. We also inferred BI to concatenated sequences tree (18S SSU and gGAPDH) using MrBayes v3. 2. (18) For this, we used GTR+I+G substitution model with 100. 000 generations with trees sampled every 10. 000 generations using chains, and 25% of the early sample trees were discarded. Historical research of deposits of Protist Collection -For each COLPROT deposit used in this work, a historical survey was done through revising the Collection database, research on abstracts of conferences and, whenever possible or necessary, interview with the depositors.
Four distinct outcomes arose from the present data: (i) isolate certified, i. e. , the DNA sequencing of the available isolate matched the sequences available in Genbank and the available literature [Supplementary data (Table  I )], (ii) isolate classification, i. e. , the isolate was not assigned to species yet, (iii) isolate re-classification, i. e. , the isolate was previously assigned to a species before the "molecular era" and it was reassigned to another taxon to better reflect the phylogeny, and (iv) new typing unit, when the DNA sequencing and the phylogenetic analysis support for a new genus/species proposition [Supplementary data (Table  I )]. Due to extensive notes of data, we comment below only the taxa that deserves highlight or the description of their taxonomic historical assembly.
Crithidia spp. Classically, this genus would be easily distinguishable from another insect trypanosomatids through morphology. The choanomastigote morphotype is advocated to be easily recognisable in light microscopy, even for non-specialised observer. However, phylogenetic studies using SSU rRNA and gGAPDH clearly indicate that this genus is polyphyletic and together with the genus Leptomonas need a revision and reclassification, as clearly demonstrated in Figure. (12, 14, 23, 24, 25) olecular phylogenetic analysis by maximum likelihood (ML) and Bayesian (BI) inferences from concatenated data set. Phylogenetic tree of isolates studied in this work and Genbank sequences inferred by ML and BI analyses using V7/V8SSU rRNA region + gGAPDH concatenated set, with General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (six categories (+G, parameter = 0.
Crithidia acantocephali and Crithidia flexonema - The type strain for C. flexonema was deposited at ATCC (50211) by LS Diamond in 1960. The trypanosomatid was isolated in Ames, IA, USA, from the host Gerris remiges,  (26)  which was later renamed to Aquarius remiges [Supplementary data (Table  III )]. (27) COLPROT016 is a duplicate. Up to now, there was no reference sequence in Genbank for this species. Another isolate without reference sequence in Genbank is C. acantocephali,  (28)  which was isolated from Acanthocephala femorata [Supplementary data (Table  III )], and deposited and preserved in ATCC, for which COLPROT042 is a duplicate. This, together with a previously unidentified deposit (COLPROT254) isolated in Brazil from Zelus sp. , present high sequence identity to COLPROT016 [Supplementary data (Table  I )] and formed a monophyletic group, as seen in Figure.
This isolate was afterwards named C. mellificae strain SF, and the whole genome was sequenced. (30) Later, Cepero research group,  (31)  while analysing honeybee colonies in Spain, compared the available gGAPDH sequences of C. mellificae isolates to the ones generated under their analysis, including C. mellificae ATCC 30254. It is clear that confusion was generated due to the deposit in Genbank of DNA sequences assumed to belong to C. mellificae without a molecular signature for the reference strain. This issue was clarified by Cepero  (31)  and Schwarz  (32)  research groups, who independently and almost simultaneously sequenced the available presumed C. mellificae strains, as well as the old available C. mellificae ATCC strains. Both authors identified considerable differences between the earlier described taxa and the more recent description of a trypanosomatid from honeybees.
abscondita (COLPROT677), C. brachyflagelli (COLPROT669), C. brevicula (COLPROT110 and COLPROT111), C. insperata (COLPROT670), C. otongatchiensis (COLPROT628 and COLPROT673) and C. permixta (COLPROT671) showed very low sequence divergence from the Genbank reference strains using the BLASTn tool and through the support of the topology observed by the phylogenetic analyses inferred by ML and BI. In this sense, these deposits had the taxonomic status confirmed [Figure  and Supplementary data (Table I)].
In this sense, Leptomonas pessoai description is full of uncertainty, the deposit COLPROT276 had few records, but the work of Galvão  (55)  that described L. pessoai was cited in the COLPROT records. By this information and by the DNA sequencing analysis, this available isolate, if actually related to the original description of Galvão, should be synonymised to H. muscarum, while the taxonomic validation of L. pessoai still waits confirmation. (11) rpetomonas samueli -The hapantotype culture is ATCC 30971 of which culture TCC 003E, deposited at the TCC-USP, and COLPROT074 are duplicates. According solely to Leptomonas lactosovorans Genbank sequences, this species should be considered a sister taxon of H. samueli. (13) L. lactosovorans (COLPROT023) is a duplicate of ATCC 30970, the known culture of the isolate described by Manaia.
(56) DNA sequencing allowed the comparison between sequences in Genbank by using the BLASTn tool. This search returned that L. lactosovorans (COLPROT023) showed high identity with the H. samueli TCC 003E isolate for the gGAPDH target [Supplementary data (Table  I )]. The phylogenetic analyses inferred by the ML and BI methods of this marker showed very robust topology values for this monophyletic clade that still includes the Leptomonas wallacei (COL-PROT194) species (Figure). (57) This last one, was isolated from Oncopeltus fasciatus raised in an insect laboratory colony [Supplementary data (Table  III )]. Herpetomonas samuelpessoai  (19)  -Herpetomonas anglusteri, which is also available at ATCC (50304), was deposited at Coleção de Tripanossomatídeos (CT-IOC) in 1992 directly by Maurilio Soares (COLPROT059).
Kentomonas sp. Kentomonas sorsogonicus -This new genus was described by Votýpka and collaborators,  (59)  from a sample isolated from Sarcophaga sp. (Diptera), collected in the province of Sorsogon (Philippines). The flagellate was described as Kentomonas and only one species was identified: K. sorsogonicus. COLPROT received a deposit of this species (COLPROT696) by Dr Vyacheslav Yurchenko in 2015. This deposit presents a sequence identical to that deposited in GB (KM242072) by the analysis of the gene gGAPDH gene, which allowed the vouchering of the new species to the institutional collection [Figure  and Supplementary data (Table I)].
Leptomonas acus, Leptomonas spiculata and Leptomonas tarcoles -COLPROT672, deposited by Dr VY Yurchenko as an isolate of L. tarcoles, shares identical gGAPDH and V7/V8 sequences to Genbank deposits of L. tarcoles (EF546787 and EF546786, respectively), which is expected [Supplementary data (Table  I )]. However, it is not possible to differentiate it from L. acus and L. spiculata solely based on gGAPDH and V7/V8 (data not shown). (23, 61) Although ecological and biological data support differences between these isolates, there is strong phylogenetic evidence supporting a careful revision of the species currently allocated in this genus. Additional loci should be formally incorporated into the identification of these complex of cryptic species, since gGAPDH and V7/V8 genes do not have resolution enough.
(62)  Therefore, we assigned it as an isolate of L. podlipaevi. Leptomonas pyrrhocoris -L. pyrrhocoris is a cosmopolitan species that mainly has the family Pyrrhocoridae as host  (64)  [Supplementary data (Table  III )]. COLPROT026 is a duplicate of ATCC 30974, which is the oldest isolate available in culture and represents the species designation. COLPROT026, COLPROT572 and 'type sequences analyses' from Genbank JN036651 (gGAPDH) and JN036653 (V7/V8) for L. pyrrhocoris  (64)  possess no nucleotide variation. COLPROT706, which has been recently isolated from Dysdercus ruficollis [Supplementary data (Table  III )], shows little or no genetic divergence for the gGAPDH and V7/V8 markers, respectively [Supplementary data (Table  I )]. Collectively, this confirms the widespread distribution of this species around the world.
(64) ptomonas seymouri -L. seymouri,  (65)  the COL-PROT277 deposit which is a duplicate of ATCC 30220, was isolated in 1959 from Dysdercus suturellus (Hemiptera: Pyrrhocoridae) in Florida, USA [Supplementary data (Table  III )]. (65) The molecular identity of this deposit was confirmed when compared to the sequence of gGAPDH (KP717896) deposited in Genbank [Figure and Supplementary data (Table  I )]. In addition, another strain isolated from D. ruficollis, collected in Rio Claro (Brazil) [Supplementary data (Table  III )] and deposited by Dr Jan Votýpka of Charles University (Prague) (COLPROT705), was also attributed to this taxon [Fig-  ure  and Supplementary data (Table  I )].
The work describes a series of isolates, which were designated a number followed by the letter T. The most studied is 9T (COLPROT189, of which COLPROT174 is a duplicate). As such, we suggest that this isolate should constitute the type strain. In 2014, COLPROT received a strain, as part of a collaboration work with Dr Vyacheslav Yurchenko. This is also a duplicate from isolate 9T, and the molecular identification confirmed the identity [Supplementary data (Table  I )]. The 9T was isolated from a tomato fruit in Rolandia, Paraná State, Brazil, the 10T isolate in Palmital, São Paulo State, Brazil, while 15T derives from an experimental infection of the insect. Although we can find citation to the 30T isolate in some old articles,  (73)  we could not track its origin exactly. Brazil and colleagues have been reporting the isolation of trypanosomatids from tomatoes and the feeding insect Phthia picta for many years, however without suc-cess in the isolation and in long-term culture of the isolates.
After they succeeded in establishing stable cultures, the authors described the isolates as Trypanosomatidae sp. (COLPROT245 and COLPROT247), due to the lack of an available culture for comparison and to the uncertainties that morphological characterisation plus host origin information already posed. (71) The present data clearly assigns COLPROT245 and COLPROT247 as strains of P. serpens, since virtually no sequence divergence was detected in both markers [Supplementary data (Table  I )]. COLPROT080 was isolated from the fruit bergamot (Citrus bergamia) by Conchon and colleagues  (74)  and deposited in 1992 in COLPROT by Marta Teixeira. This isolate had the ITS sequenced,  (10)  which assigned it to the P. serpens PhyA group. However, the authors did not generate V7/V8 or gGAPDH sequences.
(77) The cultures available nowadays refer to the isolate described by Wallace and Johnson,  (78)  as a new genus, Blastocrithidia, isolated from Aedes vexans. It was recently reclassified as a member of the Strigomonadinae subfamily under the genus Strigomonas. (12) The latter was raised to accommodate trypanosomatids that contain an endosymbiotic bacterium, which is a trait of the group, as well as their monophyletic relatedness. (12) An aposymbiotic strain was generated by prolonged antibiotic-treatment by Chang,  (79)  and it is available at COLPROT034 and ATCC. Intriguingly, Teixeira and colleagues  (12)  list ATCC 30257 as a duplicate of TCC012E, however, according to our records and ATCC catalogue, ATCC 30257 refers to the aposymbiotic strain [Supplementary data (Table  I )]. Strigomonas oncopelti -The hapantotype culture is ATCC 12982, of which culture COLPROT055 is a duplicate, as well as TCC 043E.
New genera Finally, there are three deposits, COLPROT100, COLPROT101 and COLPROT728, which do not fit in any previously described clade. COLPROT100 and COL-PROT101 were isolated from Psychodopygus ayrozai (Diptera), by Toby Barret in Brazil in 1993 [Supplementary data (Table  III )]. COLPROT728 was isolated from Oncopeltus fasciatus colony (Hemiptera) by Alexandre Romeiro in 1998. The colony was established by Dr N Ratcliffe, Swansea University-UK and further maintained by Dr Patricia Azambuja Penna, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. This insect colony is still maintained, which prompted us to screen O. fasciatus insects. As expected, the trypanosomatid infection was confirmed, and re-isolation of the trypanosomatid revealed that the molecular identity of this new isolate (COLPROT751 and COLPROT755) matched an old deposit done by Dr A Romeiro, which represented another isolate from the same host (COLPROT728).
In Conclusion -Taxonomic changes and updates are followed by "non-taxonomists" researchers with skepticism. There are long delays in changes of species names, reassemble, joining or reclassification in publications. Here, we provided a list of validated species with traceable histories and associated DNA sequences (using BLASTn tool) together the ML and BI analyses. We thereby contribute to the overall taxonomy and biodiversity knowledge of trypanosomatids and provide support for non-taxonomist researchers to clearly depict the taxonomic and phylogenetic relatedness of each sample described. This allows to correlate new isolates to the previously described ones easily and quickly. In addition, we carefully update the taxonomic information in each Genbank submission, in accordance to what was described in the present report.
It is well known by the scientific community that old or inaccurate taxonomic description of DNA sequences in Genbank reduce the pace of phylogenetic and taxonomic studies. Remarks: This isolate was originally described as Herpetomonas dedonderi by Dedet, Geoffroy & Benichou 1986, and a duplicate of the type culture was deposited in COLPROT by Erney Camargo in 1991. Teixeira  (45)  and Borghesan  (13)  suggested that this species does not fit in the genus Herpetomonas. Phylogenetic analyses conducted in the present work indicated the closer relationship with the genus Zelonia.
ACKNOWLEDGEMENTS To all the members of our laboratories for helpful and stimulating discussions. We acknowledge the use of research infrastructure of de Duve Institute that received  Dr d'Avila-Levy  for a sabbatical year, particularly  Dr Miikka Vikkula  for stimulating discussion. We are thankful to all researchers that contributed to enrich COLPROT catalogue, as well as  Fundação Oswaldo Cruz (FIOCRUZ)  for research infrastructure.
The human embryonic kidney HEK-293 cells (ATCC number CCL-1573) were grown at 37 °C with 5% CO 2 in tissue-culture flasks with vented Viruses. We used two DENV-1 strains isolated from DF cases: DENV-1 1806 (genotype V) from an autochthonous case from Nice, France in 2010 (provided by the National Reference Center of Arboviruses, France) and DENV-1 30A (genotype I) from a patient in Kamphaeng Phet, Thailand in 2010 (provided by the Afrims, Thailand and under accession number HG316482 in GenBank). The 2nd passage of DENV-1 1806 on African green monkey kidney Vero cells  60  and the 2nd passage of DENV-1 30A on C6/36 Ae. albopictus cells  61  were used for mosquito infections. Serial dilutions were used to determine the titer of viral stocks that was expressed in focus-forming units (FFU)/mL.
Sequencing libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, USA), multiplexed and sequenced in single end in two independent runs on an Illumina NextSeq 500 platform using a mid-output 150-cycle v2 kit (Illumina). Reads were trimmed (Trimmomatic v0. 33)  63  after demultiplexing (bcl2fastq v. 2. 15. 0, Illumina) to remove adaptor sequences, and reads shorter than 32 nucleotides were discarded. Full-length genome of the DENV-1 1806 was assembled de novo using Ray v2. 0. 0  64  with the original stock sample. The newly assembled DENV genome contig was extended in 3′ and 5′ using closest BLAST hit full DENV-1 genome (accession number EU482591). This chimeric construct was used to map reads used for assembly using Bowtie 2 v2. 1. 0  65. Alignment file was converted, sorted and indexed using Samtools v0.
1. 19  66. Sequencing depth was assessed using bedtools v2. 17. 0  67. Single nucleotide variants and their frequency were called using LoFreq* v2. 1. 1  68  and used to correct the chimeric construct. Only nucleotides with > 10X coverage were conserved for generating the consensus sequence. A final full-length genome sequence for DENV-1 1806 strain was deposited to GenBank (accession number MG518567). After quality control, reads from all samples were mapped to the newly assembled DENV-1 1806 strain genome sequence or previously sequenced reference genome KDH0030A (accession number HG316482) using Bowtie v2. 1. 0  65. The alignment file was converted, sorted and indexed using Samtools v0. 1. 19  66 , and the sequencing depth was assessed for each sample using bedtools v2. 17. 0  67. Single nucleotide variants (SNVs) and their frequency were then called using LoFreq* v2.
1. 1  68  , with the built-in SNV filtration using the default parameters, and their effect at the amino-acid level was assessed by SNPgenie v1. 2  69. RNA structure modeling in silico. The Mfold Web server was used with standard settings and flat exterior loop type  70  to fold the secondary RNA structures, which were then visualized using the VARNA RNA editing package  71. Pseudoknot RNA interactions were drawn as previously described for DENV  45, 72. Mutation frequencies of individual nucleotides were determined by averaging the nucleotide allele frequency from the deep sequencing results of the duplicates per treatment.
Relative fold ≥ 1. 5 is marked in bold. SD, standard deviation calculated from four replicates. A Assessment of the transcriptional organization was predicted using DOOR: a database of prokaryotic operons accessed via http://161. 117. 81. 224/DOOR3/index. php (Dam et al. , 2007; Mao et al. , 2009). The complete genome of D. dadantii strain 3937 (Genbank accession NC_014500)  (Glasner et al. , 2011)  was used as a reference. the protocol provided by the manufacturer. Southern blot transfer of bacterial genomic DNA digested with PstI restriction endonuclease was performed according to  Sambrook et al. (1989). A 679-bp PCR product of the gusA gene was used as the hybridization probe as described earlier  (Czajkowski et al. , 2017)  and the hybridization and detection were performed according to the protocol of the digoxigenin DNA-labeling and detection kit (Roche Diagnostics GmbH).
Primer sequences are shown in Supplementary Table  3. The relative, average expression levels of the candidate genes in the five biological replicates per treatment were calculated using DDCt method  (Livak and Schmittgen, 2001). The expression levels of the candidate genes were normalized with the use of the expression levels of the two reference genes lpxC and yafS  (Hommais et al. , 2011). To statistically analyze the qRT-PCR data the following statistical tests were done: the Dixon's Q test (p < 0. 05)  (Dean and Dixon, 1951)  was used for identification and rejection of outliers. The levels of expression for treatment transcripts were scaled in order to obtain an average level of expression in the reference group equal to 1 (100%). The Shapiro-Wilk test (p < 0. 05)  (Shapiro and Wilk, 1965)  was applied to test normality of results distribution for individual transcripts.
The homogeneity of variance was verified using the Fisher-Snedecor test  (Box, 1953)  or the Ansari-Bradley  (Lepage, 1971 ) test, if normal distribution was not followed. The mean expression levels for each treatment sample and the appropriate reference sample were compared using the two-tailed Student's t-test (p < 0. 05)  (Student, 1908)  when both groups were normally distributed. Otherwise, the Mann Whitney-Wilcoxon test  (Wilcoxon, 1945)  was used. Transcriptional Organization, Biochemical Pathways, and Cellular Enzymatic Networks Affected by the D. solani Tn5 Insertions The transcriptional organization (transcript of the individual gene vs. transcript as a part of the operon) was predicted for 13 D. solani IPO2222 genes containing Tn5 insertions. These predictions have been done with the use of DOOR: a database of prokaryotic operons accessed via http://161.