dataesr/scientific-paragraphs-categorization huggingface dataset:
Database Search for Protein Identification Tandem MS data generated were converted to MGF peak lists via DataAnalysis v4. 0 SP4 (Bruker Daltonics). Peak finder (sum peaks) was set to exclude any ions with <2 S/N and <1000 counts intensity. The MS/MS averaging option was unchecked, while MS and MS/MS charge deconvolution were set between 50 and 2200 m/z up to a maximum of 5+ for MS and 3+ for MS/MS spectra. The MGF peak list of each MS/MS run was imported to Proteinscape v3. 0 (Bruker Daltonics) and combined to generate a single MGF list. Protein identification was performed by searching the combined MS/MS data on a local Mascot server v2. 3 (Matrix Science) against a P. tricornutum protein database available at the Joint Genome Institute website (http://genome. jgi-psf. org/Phatr2/Phatr2. download.
ftp. html) containing 58,345 entries. Then, a single protein list was generated from all LC-MS/MS runs in separate processes. The search parameters used were as follows: enzyme specificity, trypsin/with no Pro restriction; maximum missed cleavages, 1; carbamidomethyl (Cys) as fixed modification; Deamidated (NQ), Gln->pyro-Glu (N-term Q), oxidation (Met), as variable modifications; precursor ion mass tolerance, 0. 05 D; and MS/MS mass tolerance, 0. 1 D. All data had a mass accuracy average of 4. 1 ppm. Proteins were validated statistically based on the score of their individual peptides. Proteins with at least one top-rank unique peptide with expect value below 0. 01 and ion score above 10 were accepted. Using these criteria, the final estimated false positive rate based on the decoy database search was far below 1%.
Quantitative Analysis Quantitative analysis of MS data of each fraction was performed using the Bruker's ProteinScape v3. 0 combined with ProfileAnalysis v2. 0 software (both from Bruker Daltonics). Where quantitative data were obtained for the same protein in multiple fractions, the ratio measurements from all fractions were listed. For MS data of each fraction, the data were transformed into a tabular format using a so-called bucketing approach. Each LC-MS data point was described by its retention time (RT), m/z value, and intensity. During bucketing, the extracted ion chromatography mass window and time window were assigned by automatic time alignment function, and pairs of RT-m/z values were formed and intensities assigned to each bucket (= bucket value). In order to remove systematic errors from the data set and ensure the comparability between different samples, the bucket values were normalized using the sum of all buckets.
The intensity ratios of proteins within the 95% confidence interval (mean plus minus two standard deviations) were considered to be unregulated. Proteins with a fold change out of the 95% confidence intervals (at least 2. 0-fold change in expression) were regarded as significantly regulated. Functional Annotation, Classification, and Enrichment Analysis BLAST search, mapping, and annotation of proteins differentially expressed in 36 and 60 h cultures were performed using Blast2GO software (http:// www. blast2go. de) (Conesa et al. , 2005). Functional enrichment analysis of differentially expressed proteins was performed to determine the significantly enriched GO terms and relevant proteins using BINGO 2. 44  (Maere et al. , 2005)  plug-in in the Cytoscape platform  (Shannon et al. , 2003).
Antibody Production Polyclonal antibodies of BCAT2 and BCKDH1 were generated against synthetic peptides CRTGEIVTPSLDRG and CTQLHDHLSKYPNEY. To produce antibodies against BCAT3, MCAT, and MCC2, the full-length cDNA of bcat3 and mcat, and partial cDNA sequence of the mcc2 gene, were amplified using corresponding primers (Supplemental Data Set 8) and cloned into the BamHI-XhoI sites of expression vector pGEX-4T (Pharmacia). Each expression vector was then transformed into Escherichia coli strain BL21 (DE3). For expression, a sterile 1 M solution of isopropyl-b-D-thiogalactopyranoside was added by 1:2000 dilution into a logarithmically growing bacterial culture and cells were harvested after 4 h of induction at 37°C. Following purification of antigen, immunization and sampling of the antisera from rabbit were performed by a commercial facility (Wuhan Anbiotech).
Immunoblotting Protein samples (10 mg) were dissolved in SDS-PAGE sample buffer and separated on 12% SDS-PAGE gels and then were transferred to a polyvinylidene fluoride membrane. The membranes were incubated for 1 h with a 1:500 dilution of antibodies at room temperature, followed by 1 h incubation with a 1:2000 dilution of horseradish peroxidase-labeled goat anti-rabbit IgG (Jiangsu Beyotime). Antigen-antibody complexes were visualized using the DAB horseradish peroxidase color development kit (Jiangsu Beyotime). Finally, the immunoblots were scanned, and densitometric analysis was performed using the public domain NIH Image program ImageJ (developed at the U. S. National Institutes of Health and available at http://rsb. info. nih. gov/ nih-image/). Immunoblots were performed in three independent experiments and bands of interest analyzed by ImageJ were expressed as mean 6 SD.
Construction of the RNAi Vector The vector for inverted repeat silencing constructs was generated using standard molecular cloning procedures  (Sambrook et al. , 1989). A 238-bp fragment (corresponding to the mcc2 gene sequence from 238 to 475 bp) and a 427-bp fragment (corresponding to the mcc2 gene sequence from 238 to 664 bp) were amplified from the P. tricornutum cDNA, respectively, with the primers mcc2_fw (containing a EcoRI site) and mcc2_rv1 (containing a XbaI site), and mcc2_fw and mcc2_rv2 (containing a XbaI site) (Supplemental Data Set 8). These two fragments had the first 238 bp in common. The fragments were digested with EcoRI and XbaI and ligated in sense and antisense orientations to the EcoRI site of the linearized phir-PtGUS vector  (De Riso et al. , 2009). The nat1 gene (conferring resistance to the antibiotic nourseothricin) was amplified by PCR from the pFcpE pro -NAT-FcpA39 vector and was checked by sequencing.
Biolistic Transformation The phir vectors were introduced into P. tricornutum by microparticle bombardment using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad)  (Falciatore et al. , 1999). For selection of transformants, bombarded cells were plated onto 50% fresh seawater agar plates (1% agar) supplemented with 300 mg mL 21 nourseothricin (Werner Bioagent). After 3 weeks of incubation in white light (;75 mmol photons m 22 s 21 ; 12-h photoperiod) at 20°C, individual resistant colonies were inoculated into liquid f/2 medium with 150 mg mL 21 nourseothricin. The transformants were screened by checking the integration of the nat1 gene with the primers nat1_fw and nat1_rv (Supplemental Data Set 8).
5 min. The first increment of a 2D-1 H, 1 H-NOESY pulse sequence was used for the acquisition of 1 H-NMR data and for suppressing the solvent signal. Experiments used a 100-ms mixing time along with a 990-ms presaturation (;80 Hz gB 1 ). Spectral analysis was done using Chenomx NMR suite software version 7. 7. A total of 49 metabolites were identified and quantified, and principle component analysis was applied to the metabolites' concentration data to visualize inherent clustering between wild-type and two RNAi lines.
After 10 to 12 d of incubation in white light (;75 mmol photons m 22 s 21 ) at 20°C, individual resistant colonies were inoculated into liquid f/2 medium with 40 mg mL 21 zeocin. The transformants were screened by checking the integration of the sh ble gene with the primers ble_fw and ble_rv (Supplemental Data Set 8).
Gene expression levels were normalized to the expression of the histone H4 gene. Error bars represent SE of triplicate technical replicates of duplicate cultures (n = 2). (C) Immunoblotting analysis of protein levels of BCAT2, BCAT3, BCKDH1, and MCC2 at 36 and 60 h. Cell lysate aliquots were made and normalized by intracellular protein concentration. Fold increases of protein levels from 36 to 60 h, determined using Image J software, are also shown. Normalization is shown in Supplemental Figure6.
ACKNOWLEDGMENTS This work was supported by the  National Natural Science Foundation of China  ( 40976079 ), the  National Key Basic Research Project of China  ( 2011CB200901 ), and the  Chinese Academy of Sciences   Visiting Professorship for Senior International Scientists  ( 2013T1S0004 ). F. G. acknowledges support from the  Hundred Talents Program of the Chinese Academy of Sciences. C. B. acknowledges the  EU   Micro  B3 project, as well as the  ERC  " Diatomite " and  ANR "DiaDomOil"  projects for financial support. We also thank  Jie Xiong  for help in drawing EST expression frequency figure.
2 ). A total of 13,517 protein-coding genes were annotated, underscoring that loss of gene function is not driving the small genome of B. antarctica. On the basis of a cox1 sequence data, our sample clusters with other samples collected at the same location (Supplementary Fig. 5 ). Of the 13,517 gene models, 12,914 gene models were supported by at least one RNA-seq read, and 9,011 models were supported by at least 100 RNA-seq reads. Among the annotated genes, 8,575 genes have unique alignments to entries in the SwissProt database, and 10,557 genes have matches in the non-redundant database. Genes are clustered in regions of relatively high GC content (GC content of coding regions is 47%, compared with a 37% GC content for the non-coding, Supplementary Table  8 ). We compared the B. antarctica genome with that of four other dipteran species, three mosquitoes, Ae.
De novo genome assembly. Genome size determinations from flow cytometry were produced following procedures described in Hare and Johnston  41  (see also Supplementary Methods). The assembly individual was sequenced to over 100-fold coverage using one lane of Illumina HiSeq2000 sequencing technology with a 400-bp insert paired-end sequencing library. Filtered sequence reads were assembled using Velvet de novo  15. Two iterations of ERANGE  42  were used to scaffold the assembled contigs with RNA-sequencing data from pooled larvae 10 (Supplementary Methods). Filtered sequence reads were mapped back to the reference genome using Burrows-Wheeler Aligner (bwa) with default parameters  43. PacBio scaffolding was accomplished using PBJelly (Jelly 14. 1. 14 with blasr 1. 3. 1)  44. Mapped sequence reads to the assembled genome were also used to estimate the percent PCR duplicates in the sequencing library using Picard MarkDuplicates tool  45  and to estimate coverage using BamTools  46.
gambiae TE libraries and (iii) a dipteran repeat library constructed from the 12 Drosophila genomes and An. gambiae (RepBase version 17. 08 09-01-2012, http://www. fruitfly. org/data/ p_disrupt/datasets/ASHBURNER/D_mel_transposon_sequence_set. fasta version 9. 41)  51. Species-specific TE reconstruction was attempted with ReAs  52. The R1 and R2 TE reconstruction attempt was a multi-approach attempt. Known R1 and R2 elements were downloaded from NCBI, and BLAST was used to map B. antarctica raw reads to the available R1 and R2 element sequences. Few reads mapped to the rDNA from other species, and with no significant evidence for rDNA sequence. Moreover, we undertook a targeted reassembly of the rDNA region using rDNA sequence from D. melanogaster and An. gambiae. Raw reads were mapped to the D.
melanogaster and An. gambiae 18S and 28S rDNA sequence. All reads were used in a targeted reassembly of the region, which was compared with the reference assembly and an assembly from RNA-seq data obtained from Teets et al. 10  using Trinity de novo  53. All genomic reads were then mapped to the rDNA region using bwa  43  and visually inspected for evidence of insertions or reads with split-mappings of OEA mappings that would be indicative of TE insertion. Gene annotation. Gene annotation was accomplished using the MAKER annotation pipeline  54  to map protein homology data, expressed sequence tag evidence and ab initio gene predictions to the draft genome. Protein homology data were provided by protein databases of D. melanogaster and Ae. aegypti obtained from FlyBase  55  and VectorBase  56  , respectively.
To avoid spurious matches to repetitive regions of the genome, RepeatMasker was used to mask low-complexity regions  48. In addition to the included libraries, a custom repeat library for use with RepeatMasker was created with RepeatModeler, RECON, RepeatScout and TRF  57. Filtered RNA-sequencing reads from Teets et al. 10  were mapped to the repeatmasked genome with Bowtie and TopHat and putative transcripts were assembled with Cufflinks  58. The putative transcripts were used in MAKER as expressed sequence tag evidence. An iterative approach with four rounds of training was used with MAKER and the training of the ab initio predictors SNAP  59  and AUGUSTUS  60. The benefit of the iterative approach is that with each round, the gene models improve and consequently training the ab initio predictors improves.
For the first round, SNAP was not used and the included 'fly' hidden Markov model was used in AUGUSTUS. In subsequent rounds, gene models predicted in the previous round of MAKER were used to generate hidden Markov models for SNAP and AUGUSTUS. Functional annotation was accomplished with Blast2GO  29, 61. Transcripts predicted by MAKER were compared using BLAST with the SwissProt database 62 (Eo10 À 10 ) and the non-redundant database (Eo10 À 10 ). Transcripts predicted by MAKER and the top 19 BLAST hits (blastx, NR, e-value o0. 0001) were loaded into Blast2GO. GO terms were assigned based on BLAST hits and InterProScan results  63. The set of core eukaryotic genes in the assembled genome was identified using CEGMA  64. Nuclear transfer RNA were predicted using tRNAscan-SE 1. 3. 1 (ref. 65) using options -H and -y and Aragorn 1.
2. 34 (ref. 66) using options -w -t -i116 -l -d (Supplementary Data 3). Comparative analyses. For comparative analyses, we used the first annotated isoforms for each locus from the annotations of An. gambiae (12,669)  22, 67  , Ae. aegypti (15,996)  21  , C. quinquefasciatus (18,954)  23  and D. melanogaster (13,492)  55. Clusters of orthologous genes between the five species were identified using the OrthoMCL package  27. Effective number of codons, for codon usage estimates, was calculated using ENCPrime using the implementation of Nc' that accounts for background nucleotide composition  68. Sizes of introns and exons were calculated using in-house scripts. Functional enrichment analysis. We conducted GO enrichment analysis to determine whether any functional groups of genes were enriched in B.
Results Sequencing and annotation. The sequenced cavefish individual was the first-generation offspring of two wild-caught parents, which originated from Pachon cave, Tamaulipas, Mexico. While there are at least 29 caves that contain Astyanax cavefish, the Pachon cavefish are the most studied and exhibit the most extreme troglomorphic phenotypes relative to the other caves  2. This genome draft was assembled to a size of 964 Mb, which is similar in size to a congeneric in Brazil  17. The draft genome contains 10,735 scaffolds (N50 contig ¼ 14. 7 kb; N50 scaffold ¼ 1. 775 Mb), and the longest scaffold size was 9. 823 Mb (Supplementary Data 1). Using the Ensembl annotation pipeline  18  and RNAseq transcript evidence (eight unique tissues; Supplementary Data 2), we predicted a total of 23,042 protein-coding genes, similar to other sequenced teleost fishes.
Similarly, zebrafish experienced a recent large expansion of repeat families, including Tc-Mariner and hAT superfamilies, whereas another common model, stickleback, has not (RepeatMasker Genomic Datasets). We estimated the potential age of the different of copies for each TE-related superfamilies by calculating Kimura distances assuming that most of the mutations in TE copies are neutral. The rates of transversions (q) and transitions (p) were transformed in Kimura distances using [K ¼ À ½ ln(1 À 2p À q) À 1 4 ln(1 À 2q)]. The cavefish genome differs in comparison with zebrafish in that it appears to lack very young elements (as indicated by the Kimura distance from the consensus, Fig. 1 , RepeatMasker Genomic Datasets). Given the caveats of possible assembly artefacts, lack of very young elements indicates that it is unlikely that many copies of Tc-Mariner and hAT superfamilies are still.
p. f. , 24 h. p. f. , 1. 5 days post fertilization (d. p. f. ), and 3 d. p. f. RNA from each time period was extracted from 50 whole, pooled individuals and Illumina reads were generated for cavefish and surface fish pools separately. An important caveat to interpreting the gene expression data is that even early in development, cavefish eyes are smaller than surface fish eyes, and lower numbers of transcripts may reflect smaller eyes and not necessarily downregulation. The transcript sequences were also used for obtaining coding variant differences between surface fish and cavefish. Due to the enormity of defining gene to QTL associations for many troglomorphic traits, we primarily focused on the eye phenotype. Here we used expression data and integrated pathway analysis  27  to predict likely phenotypes and the genes potentially underlying those phenotypes.
p. f. We performed an enrichment test with data combined across time points and found that the QTL were enriched for genes involved in congenital anomaly of the eye, (30/1,560 relative to 159/12,040 in the total expression data set; w 2 -test with Yates correction P value o0. 034, w 2 ¼ 4. 48, odds ratio ¼ 1. 57, 95% confidence interval of odds ratio ¼ 1. 05-2. 35). Additional genes involved in eye development, function and disease were enriched in the QTL set, though not significantly so (129/1,560 relative to 921/12,040 in total data set; w 2 -test with Yates correction P value ¼ 0. 35, w 2 ¼ 0. 88, odds ratio ¼ 1. 10, 95% confidence interval of odds ratio ¼ 0. 91-1. 34). Therefore, we contend that the eye-related QTL are qualitatively enriched for eye-related genes relative to the rest of the genome, but the eye-related QTL are quantitatively more likely to contain genes associated with congenital eye defects.
Tbx2a is involved in craniofacial and pharyngeal arch development  48  , and its paralog Tbx2b is required for proper retinal neuronal formation in zebrafish  49. Tbx2a exhibits lower expression in cavefish at all time points (Po0. 07 at 1. 5 d. p. f. , Po0. 001 at all other time points) and three nonsynonymous differences between cave and surface. Only one nonsynonymous difference is putatively derived in cavefish (D401E), and such an amino acid replacement is predicted by SIFT to be tolerated. Under the second co-localizing QTL for the traits' vibration attraction behaviour, superficial neuromast number at orbit and eye size located on LG17 in Yoshizawa et al. 7  , we lacked scaffold coverage for several markers in the center of the QTL (208e, 205d and 221a; Supplementary Data 17). There are several interesting genes in this region (Supplementary Data 11), but few are as compelling as genes found on the co-localizing QTL on LG2 of Yoshizawa et al.
These genes were not enriched for 305 gene ontology accessions related to eye development or function, and similar results were obtained for ZFIN anatomical expression data and ZFIN-predicted phenotype. Transcriptome data from the eight tissues used for gene annotation and the developmental surface fish and cavefish time series were assembled using Trinity  54  for a total of 10 separate transcriptomes. Open reading frames were predicted from these assemblies using Transdecoder in the Trinity package. We constructed a BLAST database from the coding regions of zebrafish from Ensembl Genes 74 and queried this database using each of the transcripts in the longest_orfs. cds files with BLASTn. We used a strong e-value cutoff (cutoffo1E-100), and results were robust for all values we examined from 1E-20 to 1E-100.
Thus, cavefish represent a powerful system for examining the genetic bases of evolutionary change, and we expect progress in candidate gene identification will be more efficient with the addition of the genome. While a candidate gene approach has strong potential in the cavefish system, our expression analyses highlight the discoveries enabled by a pathway approach. In one example, our analysis predicted the reduced-eye phenotype in cavefish relative to surface fish by utilizing the direction of expression within eye development pathways. Notably, this result was one of the most significant phenotypes predicted as a downstream phenotypic effect from our developmental gene expression time course of whole embryos (Supplementary Data 13-16). Nonetheless, many databases, including the ones used in this study (Ingenuity Pathway Analysis (IPA)), contain only orthologues from human, mouse and rat, and approximately 89% of the genes (2013/2408) in our QTL data set contained matches in the IPA database.
This underscores the need for future iterations of these databases to include nonmammalian model species (for example, zebrafish, Drosophila) to increase homology matches and, therefore, enable pathway analysis for a large swath of organisms. We anticipate that this genomic resource will be coupled with one of the largest strengths of the system, the repeated evolution of similar cave-associated traits in independently derived cave populations  2. Crosses between fish from different caves complement and restore certain cave-derived phenotypes (for example, rudimentary eyes)  23  ; thus, at least some of the genetic changes accounting for cave-associated traits are unique to each cave lineage  9. In addition, surface populations provide a pool of standing genetic variation for the caves  58  , and the cavefish system offers an interesting system for studying adaptation in the face of gene flow 2.
Methods Source material. Source DNA was obtained from the Jeffery Lab. DNA was collected from heart, liver, spleen and gill of a single 7-year-old adult female cavefish (Pachon) using the Genomic-Tip Tissue Midi kits (Qiagen, Valencia, CA). RNA from eight tissues was extracted with RNA Lipid Midi kits and RNeasy kits (Qiagen). Animal use complied with ethical standards and was approved by The University of Maryland Institutional Animal Care and Use Committee protocol number R-12-53 to W. R. Jeffery. Genome sequencing and assembly. Using a genome size estimate of 1. 19 Gb, total raw sequence coverage of Illumina reads was B95 Â (short insert paired-end reads, 3, 8 and 40 kb mate-paired libraries, Supplementary Data 1). The combined sequence reads were assembled using ALLPATHS software  59  and the assembled coverage was 70 Â.
This draft assembly was referred to as Astyanax mexicanus 1. 0. 2. This assembly has been gap filled with a version of Image  60  , modified for large genomes and cleaned of contaminating contigs by performing a MegaBLAST  61  of the contigs against adapter, bacterial and vertebrate databases. Identifying locations of QTL in genome assembly. Many markers overlapped between previously published QTL analyses, rendering it possible to compare coarse QTL locations across replicated maps  4, 7, 8, 15, 16  , even though LG names were not consistent between studies. Markers flanking the QTL were localized to scaffolds via best BLAST hit. Briefly, a combination of 689 RAD-tag sequences, microsatellite markers and cDNAs with linkage map positions  15  were aligned to the scaffolds using BLASTn. Astyanax mexicanus has a haploid chromosome number of 25 (ref.