Additional file 3: of Genome-scale methylation assessment did not identify prognostic biomarkers in oral tongue carcinomas

Summary of additional quality assurance analyses of our dataset against other public datasets. (DOCX 733 kb

Still waiting for the goods to arrive: The delivery of human rights to the Israeli-Palestinian conflict

Probes overlapping genetic variants at single base extension sites for Infinium Type I probes. (CSV 45 kb

Additional file 8: Table S8. of Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling

Distribution of EPIC probes across the regulatory regions of individual cell types defined using ENCODE DNAse hypersensitivity data. (XLS 46 kb

Additional file 1 of Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling

Additional file 1: Table S1. Summary of probes on EPICv2, divided by probe type and Infinium design type. Table S2. Details of nv probes and their matched variant within the COSMIC census database. Table S3. Summary of control probes on EPICv2. Table S4. Discrepant probes between Illumina manifest and script to recompute sequence from Illumina manifest 'Forward sequence' and 'IlmnID'. Table S5. Summary of number of probes per a) exact-replicate, b) location-replicate and c) sequence-only-replicate probe set. Table S6. Examples of a) exact-replicate, b) location-replicate and c) sequence-only-replicate probe sets. Table S7. Lists of IlmnIDs for probes that have different types of replicate. Table S8. Lists of IlmnIDs for probes that have different types of replicate, grouped by probe set. Table S9. Matches between EPICv2 probes and probes on older versions of the microarray based on 1) probe name, 2) target location (hg38) and 3) probe sequence of sesame manifests. Table S10. Number of replicate probes within older arrays (excluding control probes). Table S11. Number and percentage of sites targeted on each chromosome for each probe category. Table S12. Distribution of probes relative to different genomic features. Table S13. Details of samples profiled on EPICv2. Table S14. Number of probes with detection p-value >0.05 per sample. Table S15. Probes with no BLAT hits. Table S16. BLAT hit locations for probes that do not map to their target location in the Illumina manifest. Table S17. Results of competitive evaluation of location replicates

Brain size and behavioral abnormalities in <i>Rps7</i> mutants.

<p>(A) A Nissl stained coronal section of a 5 month old <i>Rps7^Mtu/+</i>brain shows a thinner cortex and larger ventricles when compared to an <i>Rps7</i>+/+ littermate. (B, C) Dissected whole brains show that the cortex is reduced in size in adult <i>Rps7^Mtu/+</i> (B) and postnatal day 0 <i>Rps7^Zma/+</i>mice (C) when compared to <i>Rps7</i>+/+ littermates. (D–I) Magnetic Resonance Microscopy (MRM) was used to visualize brain development in late gestation (E18.5) <i>Rps7</i>+/+(D–F) and <i>Rps7^Zma/+</i>(G–I) embryos. Enlarged ventricles (v in panel H) were apparent in all <i>Rps7^Zma/+</i> samples (N = 3). Representative slices are shown in sagittal (D,G), coronal (E,H), and axial (F,I) views. Scale bars = 1 mm. (J) The volume of each brain region was quantitated as a percentage of total brain volume in <i>Rps7</i>+/+(black columns) and <i>Rps7^Zma/+</i>(gray columns) (N = 3). Abbreviations: Olfactory bulbs (OB), lateral ventricles (LV), cortex (Ct), septum (Sp), striatum (St), 4^th ventricle (4V), hippocampus (Hp), thalamus (Th), colliculi (Co), cerebellum (Ce). ** indicates p<0.005. (K) Assessment of working memory by measuring spontaneous alternation in a T-maze showed a significant difference between <i>Rps7^Mtu/+</i>mice and <i>Rps7</i>+/+ littermate controls (* indicates P = 0.01, N = 9). In all panels +/+ = <i>Rps7</i>+/+; Mtu/+ = <i>Rps7^Mtu/+</i>; Zma/+ = <i>Rps7^Zma/+</i>.</p

Peripheral-blood parameters appear normal with slight developmental delay in <i>Rps7^Zma/+</i> fetal liver red cell precursors.

<p>(A) <i>Rps7^Mtu/+</i> mice displayed similar complete blood count (CBC) values to their wild-type littermates with the exception of a very slightly elevated mean corpuscular volume (MCV) (* indicates p<0.05; N = 7). (B) <i>Rps7^Zma/+</i> mice did not differ significantly from their wild-type littermates in any CBC measurements (N = 5). (C) A typical example of FACS analysis of fetal liver cells. The 5 characterized erythroid cell populations, R1–R5, are boxed in pink. (D) The average values and standard deviation for each fetal liver erythroid cell population are given as a percentage of total viable cells in Rps7+/+ and <i>Rps7^Zma/+</i> samples (* indicates p<0.001; N = 6).</p

Increased apoptosis occurs in the developing CNS of <i>Rps7^Zma/+</i> mutant embryos; however, this apoptosis is reduced in <i>Rps7^Zma/+</i>; <i>Trp53^KO/+</i> embryos.

<p>(A–C) Increased apoptosis was observed in E11.5 <i>Rps7^Zma/+</i> (Z/+) coronal sections through the neocortex compared to <i>Rps7</i>+/+ (+/+) and <i>Rps7^Zma/+</i>; <i>Trp53^KO/+</i> (Z/+;p53/+), as measured by cleaved caspase-3 (CC3) staining shown in green. (D–F) Apoptosis was also relatively increased in neural tube cross-sections of <i>Rps7^Zma/+</i> embryos at E11.5 (E). (G–I) Cellular disorganization was apparent in mitotic, phospho-histone H3-positive (PH3+) cells surrounding the lumen of the neural tube in E11.5 <i>Rps7^Zma/+</i> embryos (H). (J) E11.5 <i>Rps7^Zma/+</i> (z) embryos showed no difference from <i>Rps7</i>+/+(+) or <i>Rps7^Zma/+</i>; <i>Trp53^KO/+</i>(p) in total counts of PH3+ cells surrounding the lumen of the neural tube. (K) CC3+ cell counts confirmed significantly increased apoptosis in E11.5 <i>Rps7^Zma/+</i> (z) neural tube as compared to <i>Rps7</i>+/+ (+) or <i>Rps7^Zma/+</i>; <i>Trp53^KO/+</i> (p) (* indicates p<0.001). Scale bars: in A, D, G = 100 µM with equivalent magnification across all genotypes.</p

Heterozygous mutation of <i>Rps7</i> results in visible white spotting, small body size, and tail kinking.

<p>(A) Montu (<i>Mtu</i>) heterozygous mice exhibiting a white belly spot and kinked tail were identified in an ENU mutagenesis screen. An independent ENU screen identified zuma (<i>Zma</i>) mice with similar phenotypes (data not shown). (B) Heterozygote <i>Mtu/+</i> male (blue) and female (green) mice have a significantly reduced body weight compared to wild-type male (gray) and female (red) littermates. (C) Sequencing revealed novel <i>Rps7</i> point mutations in <i>Mtu/+</i> (c.574T>G, encoding p.V156G) and <i>Zma/+</i> (c.637A>C, encoding p.Y177S) mice. Sequence traces shown are for the antisense strand. Detailed <i>Rps7</i> genomic structure information can be found at <a href="http://www.ncbi.nlm.nih.gov/gene/20115" target="_blank">http://www.ncbi.nlm.nih.gov/gene/20115</a>. (D) The predicted structural locations of mutated amino acids in <i>Rps7^Mtu</i> and <i>Rps7^Zma</i> alleles. The three-dimensional structures of RPS7 orthologs from <i>S. cerevisiae</i> (PDB ID 3U5C_H, red) and <i>T. thermophila</i> (PDB ID 2XZM_3, gray) are superimposed. The locations of the residues homologous to mouse p.V156 (*) and p.Y177 (arrow) are shown in green (<i>S. cerevisiae</i>) and in yellow (<i>T. thermophila</i>). Image generated with UCSF Chimera.</p

Melanoblast numbers are reduced in <i>Rps7^Zma/+</i> mutants.

<p>(A, B) In transgenic embryos carrying the melanoblast reporter <i>Tg(Dct-LacZ)</i>, whole mount staining showed that Dct-positive melanoblasts are significantly reduced at E10.5 in <i>Rps7^Zma/+</i>; <i>Tg(Dct-LacZ)</i> mice (A) compared to <i>Rps7</i>+/+;<i>Tg(Dct-LacZ)</i> littermates (B). The reduction is noticeably apparent over the otic region (red circle). (C,D) Whole mount staining of E14.5 <i>Rps7^Zma/+</i>; <i>Tg(Dct-LacZ)</i> embryos showed that these embryos (D) also display a reduction in melanoblasts relative to <i>Rps7</i>+/+;<i>Tg(Dct-LacZ)</i> littermates (C). The microphthalmia observed in <i>Rps7^Zma/+</i> mice is apparent in (D). (E, F) Consistent with the whole-mount observations, transverse vibratome sections through the trunk of E14.5 embryos revealed very few melanoblasts in the developing skin of <i>Rps7^Zma/+</i>; <i>Tg(Dct-LacZ)</i> mice (F) as compared to the numerous melanoblasts seen in <i>Rps7</i>+/+;<i>Tg(Dct-LacZ)</i> littermates (arrow, E). Blue punctate staining indicates positive signal in melanoblasts. In all pairs of images, <i>Rps7</i>+/+ and <i>Rps7^Zma/+</i> are at the same magnification. NT = neural tube.</p

Eye dysmorphology in <i>Rps7</i> mutants.

<p>Representative whole-mount images from one <i>Rps7</i>+/+ and two different <i>Rps7^Zma/+</i> embryos are shown at E12.5 (A–C) and 14.5 (D–F). In addition, H&E stained sagittal sections through the eye are shown for one <i>Rps7</i>+/+ and two different <i>Rps7^Zma/+</i> embryos at E18.5 (G–I). The eye dysmorphology of <i>Rps7^Zma/+</i>mutants ranges in severity from minor unilateral or bilateral uveal coloboma (E, H) to severe microphthalmia resulting in disorganized eye structures (C, F, I). Arrows in E and F mark examples of coloboma and extreme microphthalmia, respectively. Arrow in H marks abnormal folding of the retinal layers. All images are oriented with anterior up, rostral to the right. Within each age group/row, all genotypes are shown at the same magnification. Scale bars = 0.5 mm.</p