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SPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 120 kb)

Grant agreement. Summary agreement and signature page from funder. (PDF 147 kb)

Operator training for the ASSIST Study. Details of the training programme operators received prior to study start. (DOCX 73 kb)

Schedule of review of data collected in the ASSIST Study. Details of the Sponsor/TSC/IDMC reviews. (DOCX 96 kb)

STROBE PPI. Responses of patients and public to suggested design of STROBE study. (DOCX 82â kb)

SPIRIT checklist for IMox study. (DOCX 24 kb)

Site-selection tests for the mammalian opsins. The logarithm of the model likelihood is represented by lnL, the number of model parameters is represented by np and the LRT is the likelihood ratio test. The accepted site-selection model are indicated with an asterisk (*) when the M7 model of negative selection is statistically significant, or with a double asterisk (**) if the M8 model of positive selection is statically significant. All the LRT comparisons were performed assuming a significance level of 0.05. (PDF 273Â kb)

Saturation analysis. The presence of saturation in base substitution for each of the opsin gene alignment was tested by comparing half of the expected theoretical saturation index when assuming full saturation (Iss.c, critical value) with the observed saturation index (Iss). The absence of substitution saturation is verified when Iss is lower than Iss.c for a significant p-value. (PDF 107Â kb)

Phylogenetic character mapping for the ancestral reconstructions. Ancestral reconstructions of the activity pattern and sw1-sensitivity are represented in pie charts, each slice representing the probability of each state. Ancestral inferences of the orbit convergence (degrees) and visual acuity (cycles per degree) are represented by circles that change in size and shade of grey according to the character value. The opsin trees were estimated under the assumption of the branch-specific free-ratios model using the Meredith et al. (2011) tree topology. (PDF 1239Â kb)

Phylogenetic character mapping for the ancestral reconstructions. Ancestral reconstructions of the activity pattern and sw1-sensitivity are represented in pie charts, each slice representing the probability of each state. Ancestral inferences of the orbit convergence (degrees) and visual acuity (cycles per degree) are represented by circles that change in size and shade of grey according to the character value. The opsin trees were estimated under the assumption of the branch-specific free-ratios model using the Meredith et al. (2011) tree topology. (PDF 1239Â kb)

Saturation analysis. The presence of saturation in base substitution for each of the opsin gene alignment was tested by comparing half of the expected theoretical saturation index when assuming full saturation (Iss.c, critical value) with the observed saturation index (Iss). The absence of substitution saturation is verified when Iss is lower than Iss.c for a significant p-value. (PDF 107Â kb)

Venn diagrams of positively-selected bone-associated genes. The intersection between the positively selected genes in the three clades, birds, mammals and reptiles. (DOC 149 kb)

Divergence limit estimations. Calibration points retrieved from TimeTree. (DOC 35 kb)

Positively selected sites of bone-associated genes in Reptilian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Bold represents statistical significance (pâ

Bovine BAC clone localization on grey brocket deer chromosomes. Table S2. Repeat family composition. Table S3. Major repeat clusters revealed in chromosome-specific DNA with RepeatExplorer. Figure S1. Insert length distribution for chromosome-specific DNA containing bovine chromosomes 23, 26, 28 and 29) inferred from paired-end read mapping to cattle genome. Figure S2. Fluorescence in situ hybridization of chromosome-specific DNA samples containing mixed bovine chromosomes 23, 26, 28 and 29 (red - CY3) and chromosome 10 (green - FITC) to cattle metaphase and corresponding DAPI staining. Figure S4. Fluorescence in situ hybridization of bovine BAC clone CH240-472E2 to grey brocket deer metaphase and corresponding DAPI staining. (DOC 633Â kb)

Positively selected sites of bone-associated genes in Reptilian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Bold represents statistical significance (pâ

Branch-site model for birds. Genes were the alternate model was preferred relatively to the null model are highlighted as italic and underlined positively selected. (DOC 141 kb)

Skeletal adaptations to flight in bats. Skeleton of Large flying fox (adapted from Wikimedia Commons licensed under a Creative Commons Attribution-Share Alike 3.0 Unported (CC BY-SA 3.0)) and the key features observed in bats skeleton system. The typical bone structure of long bones is highlighted in the light blue box (adapted from Wikimedia Commons licensed under a Creative Commons Attribution-Share Alike 3.0 Unported (CC BY-SA 3.0)). (DOC 331 kb)

Body mass in birds and mammals. (DOCX 46 kb)

Branch model for birds. Bold represents statistical significance (pâ

Positively selected sites of bone-associated genes in Avian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Gene in bold are positively selected under the comparison M2a vs M1a. Q-value estimations for multiple testing are represented as positive selected (1) and negative selected (0). (DOC 88 kb)

Bovine BAC clone localization on grey brocket deer chromosomes. Table S2. Repeat family composition. Table S3. Major repeat clusters revealed in chromosome-specific DNA with RepeatExplorer. Figure S1. Insert length distribution for chromosome-specific DNA containing bovine chromosomes 23, 26, 28 and 29) inferred from paired-end read mapping to cattle genome. Figure S2. Fluorescence in situ hybridization of chromosome-specific DNA samples containing mixed bovine chromosomes 23, 26, 28 and 29 (red - CY3) and chromosome 10 (green - FITC) to cattle metaphase and corresponding DAPI staining. Figure S4. Fluorescence in situ hybridization of bovine BAC clone CH240-472E2 to grey brocket deer metaphase and corresponding DAPI staining. (DOC 633Â kb)

Positively selected sites of bone-associated genes in Mammalian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Gene in bold are positively selected under the comparison M2a vs M1a. Q-value estimations for multiple testing are represented as positive selected (1) and negative selected (0). (DOC 88 kb)

Branch model for mammals. Genes without flying mammals present in the alignment are marked (###). Bold represents statistical significance (pâ

Estimation of dN and dS for each branch under Model 0. For each branch, average of dN and dS and the corresponding standard deviation. (DOC 165 kb)

Genomic location of bone-associated genes. The circular ideogram represents the genomic location of bone-associated genes in four of the studied species. Each end-line represents the location of the bone-associated genes. Blue indicates human chromosomes (mammal representative). Dark orange the zebra finch (flying species), green the chicken and yellow the turkey (flightless species). (DOC 896 kb)

Positively selected sites of bone-associated genes in Mammalian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Gene in bold are positively selected under the comparison M2a vs M1a. Q-value estimations for multiple testing are represented as positive selected (1) and negative selected (0). (DOC 88 kb)

Avian and Mammalian phylogenetic trees used in CODEML analysis. Lineages of flightless birds are highlighted in red, while flying mammals are highlighted in blue. (DOC 339 kb)

Branch-site model for mammals. Genes without flying mammals present in the alignment are marked (###). (DOC 138 kb)

Supplemental Methods. Further details of species identification using mtDNA consensus mapping method; raw read filtering criteria; genome size estimation using K-mer analysis; leopard genome assembly using various K-mer values; repeat annotation; species selection for comparative genomic analysis. (DOCX 43 kb)

Results from the nested models (M0, M1a, M2a) likelihood ratio test results PAML from Mammalian dataset excluding bats. The alignment length is on Amino acids (aa). Bold represents statistical significance (pâ

Species and sub-species identification for three leopard samples. Figure S2. Distribution of K-mer frequency in the error-corrected reads. Figure S3. GC content distributions. Figure S4. Composition of mammalian orthologous genes. Figure S5. Divergence time estimation of 18 mammals. Figure S6. Contraction of the amylase gene families (AMY1 and AMY2) in carnivores. Figure S7. Frame-shift mutations in Felidae GCKR genes. Figure S8. Felidae-specific amino acid changes in DNA repair system. Figure S9. Felidae-specific amino acid change in MEP1A protein. Figure S10. Felidae-specific amino acid change in ACE2 protein. Figure S11. Felidae-specific amino acid change in PRCP protein. (DOCX 2024 kb)

Species and sub-species identification for three leopard samples. Figure S2. Distribution of K-mer frequency in the error-corrected reads. Figure S3. GC content distributions. Figure S4. Composition of mammalian orthologous genes. Figure S5. Divergence time estimation of 18 mammals. Figure S6. Contraction of the amylase gene families (AMY1 and AMY2) in carnivores. Figure S7. Frame-shift mutations in Felidae GCKR genes. Figure S8. Felidae-specific amino acid changes in DNA repair system. Figure S9. Felidae-specific amino acid change in MEP1A protein. Figure S10. Felidae-specific amino acid change in ACE2 protein. Figure S11. Felidae-specific amino acid change in PRCP protein. (DOCX 2024 kb)

Branch-site model for mammals. Genes without flying mammals present in the alignment are marked (###). (DOC 138 kb)

Venn diagrams of positively-selected bone-associated genes. The intersection between the positively selected genes in the three clades, birds, mammals and reptiles. (DOC 149 kb)

List of bone associated genes used in this study. (DOC 61 kb)

Phylogenetic trees of reptiles used in CODEML analysis. (DOC 90 kb)

Positively selected sites of bone-associated genes in Avian dataset after multiple testing correction. The alignment length is on Amino acids (aa). Gene in bold are positively selected under the comparison M2a vs M1a. Q-value estimations for multiple testing are represented as positive selected (1) and negative selected (0). (DOC 88 kb)

Genomic localization of the genes used for the co-evolution analysis. Genomic localization of the visual genes (in orange) and melanin-based plumage coloration genes (in red) in the chicken and zebra finch karyotypes. (PDF 15 kb)

Amino acid sequence of the barn owl ( Tyto alba ) RH2 opsin. RH2 conopsin multiple sequence alignment of the barn owl and the zebra finch (Neoaves representative), chicken (Galloanseres representative) and ostrich (Paleognathae representative). Regions in red indicate special features of the barn owl RH2 sequence: non-synonymous mutations, indels and stop-codons. (PDF 130 kb)

Saturation analysis. The presence of saturation in base substitution for each of the gene alignment was tested by comparing half of the theoretical saturation index expected when assuming full saturation (Iss.c, critical value) with the observed saturation index (Iss). Absence of substitution saturation is verified when Iss is lower than Iss.c for a significant p-value. (PDF 24 kb)

Avian sw2 and lw sequences. Accession number of the sw2 and lw sequences used for site selection analyses. (PDF 12 kb)

Estimation of genome size using 17-mers. Fig. S2 The divergence time of avian species. Fig. S3 Specific amino acid changes in the gastric acid secretion associated genes. Fig. S4 Species distribution of BLASTx top hits of cinereous vulture transcripts. Fig. S5 NR protein database properties for the assembled unigenes. Fig. S6 Gene Ontology classifications of cinereous vulture unigenes. Fig. S7 Amino-acid sequence comparison of toll-like receptor 1 of the Griffon and cinereous vultures. (DOCX 2228 kb)

Mammalian opsin sequences. Accession number of the mammalian opsins sequences used for site selection analyses. (PDF 88 kb)

Species-specific branch selection tests. The one-ratio model (H0) was tested against the two-ratios model considering the alternative hypotheses of verifying differentiated Ď -ratios in the terminal lineage of the barn owl (H1.1) or the emerging lineage of penguins (H1.2). Significant alternative hypothesis are marked with a red p-value. lnL is the logarithm of the model likelihood and the LRT is the likelihood ratio test. All the LRT comparisons were performed with 1 degree of freedom and assuming a significance level of 0.05. (PDF 147 kb)

GC ratio comparisons for avian opsins. The GC ratio corresponds to the %GC / [%G * %C] ratio. GC ratio equal to 1 corresponds to the absence of GC bias, while value lees than 0.76 or higher than 1.25 (horizontal red lines in the plot) indicate deviances in GC use. (PDF 12 kb)

Supplemental figures. Figure S1. Cheetah genome size estimation by 17-mers. Figure S2. Depth distribution of cheetah reads. Figure S3. GC content and average sequencing depth values. Figure S4. Depth distribution of re-sequencing reads. Figure S5. Distribution of syntenic blocks in genome windows. Figure S6. Ten largest cat-cheetah rearrangements. Figure S7. Size of homozygosity stretches in Felidae genomes. Figure S8. Ideograms of homozygosity regions. Figure S9. Comparison of cheetah and human MHC regions. Figure S10. Comparison of cheetah and dog MHC regions. Figure S11. Inferred historical population sizes by PSMC analysis. Figure S12. Bootstrap values for DaDi demographic models. Figure S13. Alignments of the AKAP4 gene. Figure S14. Evolutionary history of LDH gene families. Figure S15. Cumulative distribution of 36-mers. Figure S16. Copy-number distribution in control regions. Figure S17. Example of fixed duplications on scaffold606. (PDF 2027 kb)

UVS/VS condition for avian sw1 sequences. Avian sw1 sequences used in this study and the respective accession numbers. UVS/UV sw1 type was inferred using the 86, 90 and 93 spectral tuning sites (highlighted in red). Amino acid combinations were compared with previously published sequences in Ă deen et al. (2013) [16]. (PDF 174 kb)

Avian opsin and melanin-based plumage coloration sequences. Accession numbers are indicated for the complete sequences, while for partial sequences the genomic location is given. Asterisk (*) indicate that we were not able to identify the gene in the respective genome. Gene sequences are available in the Avian Phylogenomics Project database ( http://avian.genomics.cn/en/ , [75]). (PDF 205 kb)

Log GC ratio pairwise comparisons between the avian opsins. The presented p-values are corrected using the Bonferroni adjustment. p-values colored in red represent statistically non-significant differences between the average log GC ratios of the compared genes. Pairwise comparisons for the sw1, sw2 and lw opsins are highlighted in grey. (PDF 27 kb)