Additional file 3: of Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Table S2. Differentially expressed genes between clusters 1 and 8. (XLSX 85 kb

Additional file 6: of Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Table S5. Buffer composition for the poly-tailing step. (XLSX 33 kb

Unusually Large Number of Mutations in Asexually Reproducing Clonal Planarian <i>Dugesia japonica</i>

<div><p>We established a laboratory clonal strain of freshwater planarian (<i>Dugesia japonica</i>) that was derived from a single individual and that continued to undergo autotomous asexual reproduction for more than 20 years, and we performed large-scale genome sequencing and transcriptome analysis on it. Despite the fact that a completely clonal strain of the planarian was used, an unusually large number of mutations were detected. To enable quantitative genetic analysis of such a unique organism, we developed a new model called the Reference Gene Model, and used it to conduct large-scale transcriptome analysis. The results revealed large numbers of mutations not only outside but also inside gene-coding regions. Non-synonymous SNPs were detected in 74% of the genes for which valid ORFs were predicted. Interestingly, the high-mutation genes, such as metabolism- and defense-related genes, were correlated with genes that were previously identified as diverse genes among different planarian species. Although a large number of amino acid substitutions were apparently accumulated during asexual reproduction over this long period of time, the planarian maintained normal body-shape, behaviors, and physiological functions. The results of the present study reveal a unique aspect of asexual reproduction.</p></div

Additional file 4: of Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Table S3. Enrichment analysis of biological pathways using the genes differentially expressed between clusters 1 and 8. (XLSX 404 kb

Additional file 5: of Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

Table S4. Primer sequences. (XLSX 148 kb

FACS profiles of cells derived from <i>D</i>. <i>japonica</i> and <i>S</i>. <i>mediterranea</i>.

<p>Fluorescence-activated cell sorting (FACS) profiles of cells derived from the whole body of <i>D</i>. <i>japonica</i> SSP-strain (Dj-SSP) and <i>S</i>. <i>mediterranea</i> CIW4-strain (Sm-CIW4). The number of cells analyzed was 9,104 and 11,588, respectively. Each dot indicates the relative fluorescence intensity of Calcein AM and Hoechst 33342, and red color indicates a relatively high population of cells. Calcein AM labels the cytosol of viable cells, and its intensity is plotted on a logarithmic scale on the X-axis. Hoechst 33342 labels chromosomes, and is used to estimate the genome size, and its intensity is plotted on a linear scale on the Y-axis.</p

Information about transcriptome sequences.

<p>Information about transcriptome sequences.</p

Summary of SNP analysis.

<p>Summary of SNP analysis.</p

Statistics of transcriptome assembly.

<p>Due to the difference of the terms of the assembled sequence between Trinity and Newbler, "isogroup" is defined as "gene-group", and "isotig" is defined as "gene-isoform".</p><p>Statistics of transcriptome assembly.</p

k-mer spectra of <i>D</i>. <i>japonica</i> and control genome.

<p>(A) shows the control results for <i>S</i>. <i>venezuelensis</i>, which is known to be highly heterozygous (0.927%), and showed a bimodal peak consistent with its genome characteristics. (B) shows the results for <i>D</i>. <i>japonica</i>, and neither a monomodal nor a bimodal peak was found.</p