Additional file 1: of The practical use of genome sequencing data in the management of a feline colony pedigree

Additional tables. Table S1. Validation primer sequences and PCR annealing temperatures; Table S2. Heterozygous SNPs selected from SNPchip data for cross validation; Table S3. Variants associated with diseases in cats with a commercial DNA test available; Table S4. LoF variants identified in genes associated with disease; Table S5. Inbreeding coefficient calculated with SNPchip data of only 3 cats and with SNPchip data of 297 cats ; Table S6. RoH identified with SNPchip data on the three cats; Table S7. RoH identified with WGS data on the three cats. The last column has the overlap with SNPchip RoH number. (DOCX 42 kb

Number of unique drugs predicted to target each known medaka melanoma differentially expressed gene.

<p>The drug list is presented in full in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143057#pone.0143057.s003" target="_blank">S3 File</a>.</p

Genes known to be associated with melanocyte development and pigmentation signaling.

<p>Red and green colored gene symbols are increased or decreased, respectively, in melanoma fishes compared to control. The <i>mitf</i> gene (yellow) is upregulated but fails to reach statistical significance.</p

Outbred (pigmented) <i>B</i>. <i>glabrata</i> snails contain higher DNA methyltransferase activity, MBD-binding capacity and genome 5mC compared to inbred (NMRI) snails.

<p>DNA methyltransferase and MBD-binding activity was fluorometrically measured in nuclear protein isolated from the head/foot tissue of adult snails. <b>A) DNMT activity was measured in 7</b> μ<b>g of <i>B</i>. <i>glabrata</i> (NMRI and pigmented hybrid strains) nuclear protein extract (n = 2) using EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek).</b> Relative fluorescence units (RFU) were obtained at 530_EX/590_EM nm and subsequently normalised to the blank negative control (assay buffer only) and positive control (Dnmt1). Error bars represent standard deviation (SD) of the normalised means. <b>B) MBD-binding activity within 10</b> μ<b>g of <i>B</i>. <i>glabrata</i> (NMRI and pigmented hybrid strains) nuclear protein extract (n = 2) was measured with the EpiQuik MBD2 Binding Activity/Inhibition Assay Kit (Epigentek).</b> 10 μg of BSA was used as a negative control. Fluorescence was read at 530_EX/590_EM nm and readings subsequently normalised to the blank negative control (assay buffer only). Error bars represent ± standard deviation (SD) of the normalised means. <b>C) 5mC was detected in <i>B</i>. <i>glabrata</i> gDNA (100ng) derived from both albino NMRI and pigmented hybrid strains (n = 2) using the MethylFlash methylated DNA Quantification Kit (Epigentek).</b> The level of 5mC was measured in relative fluorescence units (RFU) at 530_EX/590_EM nm and normalised to the negative (synthetic unmethylated DNA with 50% cytosine content) and positive control (synthetic methylated DNA with 50% 5mC content). * indicates a significant difference (Student’s two-tailed t test; <i>p<0</i>.<i>05</i>) between the 5mC level of NMRI and Pigmented snails. Readings are shown as means and error bars represent ± standard deviation (SD). 5mC abundance (%), displayed above bars, was calculated based on the <i>B</i>. <i>glabrata</i> genome GC content (35%) as described in the Materials and Methods.</p

<i>B</i>. <i>glabrata</i> DNMT homologs are novel members of the DNMT enzyme family.

<p>Phylogenetic relationships based on Bayesian (Mr Bayes v3.1.2) and Maximum Likelihood (MEGA v5.2.2) approaches, were inferred from a multiple sequence alignment of the six highly conserved motifs within the catalytic domain (PF00145) from 29 taxa using MUSCLE [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref031" target="_blank">31</a>]. BgDNMT1 and BgDNMT2 are indicated by red boxes. Abbreviations Bg, Ac, Lg, Ct, Hr, Ci, Mm, Smd, Em, Sm, Fh, Am and Cq relate to <i>B</i>. <i>glabrata</i>, <i>A</i>. <i>californica</i>, <i>L</i>. <i>gigantea</i>, <i>C</i>. <i>gigas</i>, <i>C</i>. <i>teleta</i>, <i>H</i>. <i>robusta</i>, <i>C</i>. <i>intestinalis</i>, <i>M</i>. <i>musculus</i>, <i>S</i>. <i>mediterranea</i>, <i>E</i>. <i>multilocularis</i>, <i>S</i>. <i>mansoni</i>, <i>F</i>. <i>hepatica</i>, <i>A</i>. <i>mellifera</i> and <i>C</i>. <i>quinquefasciatus</i>. The Bayesian analysis consensus tree is illustrated (Figtree v1.3.1, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref036" target="_blank">36</a>]) with branch lengths signifying distance between taxa. Node labels within parentheses represent percentage bootstrap support values from Maximum Likelihood analysis (500 bootstrap replicates performed using the JTT model), while those outside parentheses represent Bayesian posterior probability support values (based on performing four independent Markov Chain Monte Carlo runs for 1,000,000 generations using the WAG model).</p

The <i>B</i>. <i>glabrata</i> 14-3-3 (BGLB005695) single exon gene contains methylated cytosines within its coding region.

<p><b>A) PCR amplification of bisulfite converted DNA reveals four 5mC sites within Bg14-3-3.</b> Bisulfite conversion of gDNA followed by PCR (BS-PCR) and sequencing was employed to investigate the methylation status of cytosines within the exon of <i>Bg14-3-3</i> (Scaffold1582:42425–42875). Following bisulfite conversion of gDNA, target sequences were amplified, subcloned, and individual clones subsequently sequenced. A filled circle indicates the presence of 5mC; an empty circle indicates the absence of 5mC. Each circle represents a cytosine in a CpG context. Numbers above the circles correspond to base pair positions. Percentages of 5mC detected at each cytosine are indicated. Grey box represents CDS of <i>Bg14-3-</i>3 and the dashed lines indicate the amplified region. B) <b>Confirmation of methylated CpGs within <i>Bg14-3-3</i> by WGBS.</b> An IGV v2.3 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref057" target="_blank">57</a>] genome browser screenshot of the <i>Bg14-3-3</i> gene. Black bars indicate a methylated CpG position as determined by WGBS (Genome Publication, under review) and y-axis represents degree of methylation (between 0–1) as estimated by BSMAP v1.0.0 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref100" target="_blank">100</a>].</p

The <i>B</i>. <i>glabrata</i> DNA methylation machinery is abundantly expressed in sex tissues and haemocytes.

<p><b>A) RNA-Seq analysis of the <i>B</i>. <i>glabrata</i> DNA methylation machinery in twelve snail tissues.</b> The normalised sequencing counts [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref051" target="_blank">51</a>] for each gene of interest (i.e. <i>Bgmbd2/3</i>, <i>Bgdnmt1</i> and <i>Bgdnmt2</i>) across the twelve tissues were used to estimate sample parameters for that gene i.e. the mean and standard deviation. The twelve observations for each gene were scaled to a standardised t-distribution. These standardised counts for the three genes were plotted (y-axis) against the twelve tissues (x-axis)—the continuous the red line on the y-axis at 1.79 represents <i>p</i> < 0.05 on a t-distribution with 11 degrees of freedom. The samples were divided into three groups, i.e Group 2: ovotestes (OVO), Group 3: terminal genitalia (TRG) and Group 1: salivary glands (SAL), digestive gland/hepatopancreas (DG/HP), central nervous system (CNS), buccal mass (BUC), albumin gland <b>(</b>AG), mantle edge (MAN), head/foot (FOOT), stomach (STO), heart/APO (HAPO) and kidney (KID). Differential expression analysis, using DESeq2 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref051" target="_blank">51</a>], indicates the Group 1 vs. Group 2 and Group 1 vs. Group 3 comparisons of <i>Bgmbd2</i>/<i>3</i>, <i>Bgdnmt1</i> and <i>Bgdnmt2</i> abundance (i.e. tissue samples with data above the red line) are statistically significant for that gene of interest. <b>B) qRT-PCR data confirms the tissue-enriched expression of the <i>B</i>. <i>glabrata</i> DNA methylation machinery.</b> qRT-PCR was employed to verify the transcript abundance of <i>Bgdnmt1</i>, <i>Bgdnmt2</i> and <i>Bgmbd2/3</i> across five tissues previously analysed by RNAseq. In addition to albumin gland <b>(</b>AG), head/foot (FOOT), stomach (STO), ovotestes (OVO) and digestive gland/hepatopancreas (DG/HP), transcript abundance was also determined in haemocytes (HAEMO). Error bars represent standard deviation of the mean (SD). The Ct values of target genes were normalised to the reference gene S19 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref077" target="_blank">77</a>]. Biological duplicates were used for each tissue and technical triplicates performed for every qRT-PCR reaction. For haemocytes, only one biological sample was available. <b>C) 5-AzaC treatment inhibits <i>B</i>. <i>glabrata</i> oviposition.</b> Adult NMRI snails (10–12 individuals/condition) were incubated in the presence or absence of 491μM 5-AzaC for a total of eight days. The bar chart represents mean eggs laid/condition at day eight + standard deviation (SD). The Student’s two-tailed <i>t</i> test was performed to identify significant differences between the treatments. Images are representative of egg sacs obtained from control and 5-AzaC conditions and were taken 7 days after deposition. <b>D) A heat map representation of genes within the neighbourhood of <i>Bgdnmt1</i> and <i>Bgmbd2/3</i> that are significantly over or under-expressed in OVO (ovotestes).</b> The genes are clustered in two directions i.e. across samples and across genes. Uniprot assigned short names to these genes based on sequence homology (full name included in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.s002" target="_blank">S2 Table</a>) are indicated.</p

<i>B</i>. <i>glabrata</i> contains a methyl-CpG-binding protein, BgMBD2/3, which shares high sequence similarity with eukaryotic MBD proteins.

<p>Alignment of methyl-CpG binding domain (PF01429) and C-terminal domain of methyl-CpG-binding domain protein 2 & 3 (PF14048) regions using MUSCLE [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref031" target="_blank">31</a>]. Abbreviations Bg, Ac, Lg, Cg, Hr, Ct, Sm, Em, Smed, Bm, Hp, and Mm refer to <i>B</i>. <i>glabrata</i>, <i>A</i>. <i>californica</i>, <i>L</i>. <i>gigantea</i>, <i>C</i>. <i>gigas</i>, <i>H</i>. <i>robusta</i>, <i>C</i>. <i>teleta</i>, <i>S</i>. <i>mansoni</i>, <i>E</i>. <i>multilocularis</i>, <i>S</i>. <i>mediterranea</i>, <i>B</i>. <i>mori</i>, <i>H</i>. <i>pulcherrimus</i> and <i>M</i>. <i>musculus</i>. An asterisk in the upper line indicates functionally important amino acids within the methyl binding domain region. Numbers at the beginning of each line represent amino acid positions and at each position the most conserved residues are further shaded in black, semi-conserved residues are highlighted grey and non-conserved amino acids are kept white. 'Consensus' represents the Pfam consensus sequence of each domain where conserved amino acids (50–79%) are indicated by lower-case and highly conserved residues (> 80%) by upper-case letters. Missing amino acid residues, not present in the truncated CtMBD2/3 candidates, are indicated by a ‘N/A’.</p

BgMBD2/3 represents a homolog of the invertebrate-specific MBD2/3 clade.

<p>Phylogenetic reconstruction based on Bayesian (Mr Bayes v3.1.2) and Maximum Likelihood (MEGA v5.2.2) methods were estimated from a multiple sequence alignment based on the MBD domain of 28 eukaryotic MBD sequences. Notations Cs, Ov, Pw, Sm, Sj, Fh, Em, Eg, Ts, Hm, Smed, Hr, Lg, Ac, Cg, Ct, Hp, Mm, Xl and Xt refer to <i>C</i>. <i>sinensis</i>, <i>O</i>. <i>viverrini</i>, <i>P</i>. <i>westermani</i>, <i>S</i>. <i>mansoni</i>, <i>S</i>. <i>japonicum</i>, <i>F</i>. <i>hepatica</i>, <i>C</i>. <i>sinensis</i>, <i>E</i>. <i>multilocularis</i>, <i>E</i>. <i>granulosus</i>, <i>T</i>. <i>solium</i>, <i>H</i>. <i>microstomum</i>, <i>S</i>. <i>mediterranea</i>, <i>M</i>. <i>lignano</i>, <i>H</i>. <i>robusta</i>, <i>L</i>. <i>gigantea</i>, <i>A</i>. <i>californica</i>, <i>C</i>. <i>gigas</i>, <i>C</i>. <i>telata</i>, <i>H</i>. <i>pulcherrimus</i>, <i>M</i>. <i>musculus</i>, <i>X</i>. <i>laevis</i> and <i>X</i>. <i>tropicalis</i>. A graphical output of the Bayesian consensus phylogram was obtained via Figtree v1.3.1 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref036" target="_blank">36</a>] and BgMBD2/3 is included within a red box. Node labels within parentheses represent percentage bootstrap support values from Maximum Likelihood analysis (500 bootstrap replicates performed using the JTT model), while those outside parentheses represent Bayesian posterior probability support values (based on performing four independent Markov Chain Monte Carlo runs for 1,000,000 generations using the WAG model). Only nodes with Bayesian posterior probability support values > 0.5 are shown.</p

The <i>B</i>. <i>glabrata</i> genome encodes both DNA methyltransferase 1 (BgDNMT1) and 2 (BgDNMT2) family members.

<p><b>A) Cartoon representation of BgDNMT1 and BgDNMT2.</b> C-terminal catalytic DNA methylase domains (PF00145) present in BgDNMT1 and BgDNMT2 are highlighted in red. Roman numerals (I, IV, VI, VIII, IX & X) within the DNMT domain denote the highly conserved motifs and TRD represents the target recognition domain. In BgDNMT1, this DNA methylase domain is linked to a N-terminal regulatory region via a KG linker (black vertical bar; amino acid position 833–843). The regulatory domain contains a nuclear localisation signal (NLS), indicated with an asterisk, a cytosine-specific DNA methyltransferase replication foci domain (RFD; PF12047), a Zinc Finger CXXC Domain (PF02008) and two bromo-adjacent homology (BAH) domains (PF01426), coloured in green, grey and turquoise respectively. Numbers represent amino acid positions. <b>B) <i>B</i>. <i>glabrata</i> DNMT homologs share extensive sequence similarity throughout the DNA methylase catalytic domain (PF00145).</b> A multiple sequence alignment of the invariant C-termini (as predicted by Pfam domain search) of BgDNMT1 and BgDNMT2 with homologous enzymes was performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation; [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005246#pntd.0005246.ref036" target="_blank">36</a>]). The six highly conserved motifs of the catalytic domain are indicated with Roman numerals (I, IV, VI, VIII, IX & X) and TRD marks the Target Recognition Domain. Functionally important residues are highlighted with an asterisk and numbers at the beginning of each motif represent the amino acid positions. The most conserved residues are further shaded in black, semi-conserved residues are highlighted grey and non-conserved amino acids are white. 'Consensus' represents the Pfam consensus sequence of each domain where conserved amino acids (50–79%) are indicated by lower-case, and highly conserved residues (> 80%) by upper-case letters.</p