1 Unidad de Genotipado y Diagnóstico Genético (UGDG), INCLIVA, Valencia (Spain) 2 Unidad de Bioinformática, INCLIVA, Valencia (Spain) 3 Hospital Universitari de Tarragona Joan XXIII / IISPV / Universitat Rovira i Virgili / CIBERDEM (Spain) Daniel Pérez-Gil1,2, Verónica Lendínez-Tortajada1, Alba Sanchis-Juan1,2, Pilar Rentero-Garrido1, Ana Megía3, Joan Vendrell3, Felipe Javier Chaves-Martínez1, Pablo Marín-García1,2 DPMAS: pipeline for 450K top table replication with MiSeq amplicons ABSTRACT DNA methylation is one of the most important epigenetic modifications in gene expression regulation and it plays a crucial role in multiple biological processes. Therefore, DNA methylation profiling is important to reveal regions of the genome that are altered during development or that are perturbed by disease. Methylation signals at CpG sites can be assessed using bisulfite conversion method and massive sequencing technologies. Here we present the implementation of a bioinformatics pipeline for replication with MiSeq sequencing amplicons of the top hits obtained from Infinium 450K differential methylation experiments. CONCLUSIONS We have implemented a bioinformatics pipeline for replication of the top hits obtained from Infinium 450K experiments using MiSeq sequencing amplicons. For this purpose we have used well-established software for genome analysis as well as applications developed in our lab, all of them open source. The proposed workflow has proved to be useful for methylation level assessment while maintaining a good data quality control. Source code will be available at: https://github.com/pamag/DPMAS PIPELINE REPORT 450K results The assessment of methylation level at CpG sites from raw genetic sequences involves quality control and several processing steps. The main building blocks of the pipeline include SNP selection from 450K experiments, sequencing of the regions containing those SNPs with directional primers, mapping of the reads to the reference amplicon sequences and variant calling at cytosines in CpG sites. Finally, a dashboard report is produced as an analysis summary. Other SNP selection Directional amplification primers design MiSeq sequencing Adaptor removal and quality trimming Mapping Min length filter Calling C and T counts REPORT Beta values plot FASTQ BAM VCF Figure 2. Coverage plot. Depth per base distribution is plotted for each sample (black lines) in every experimental group. In this example a threshold of 100x is established as a minimum value to report reliable results (red line). Figure 3. Methylation plots. Beta values plot (A). Methylation percentages of CpG sites along the amplicon are plotted for each sample in every experimental group. The 450K selected SNPs are highlighted as a red line. In this example only those samples with coverage >=100 at the CpG site are plotted. A reliability value is assigned to each point depending on the error assumed when the methylation level is assigned (B). Figure 1. Localization plot. The region where the selected 450K SNPs are located is plotted. Chromosome and gene transcripts are displayed and the CpG of interest is shown as a red line. Table 1. Methylation level table. Mean, standard error and number of samples (mean±se(N)) are shown for each CpG site in every experimental group. The CpG site of interest is highlighted in red. Position c1 c2 c3 c4 64 77.71±0.63(46) 79.03±0.49(85) 78.69±0.61(60) 78.61±1.02(62) 105 89.76±0.37(46) 89.3±0.42(85) 90.38±0.45(60) 89.82±0.6(61) 108 94.75±0.21(46) 95.01±0.24(85) 94.62±0.24(60) 94.61±0.42(62) 119 54.15±4.57(46) 52.69±3.43(85) 55.51±4.14(60) 56.16±3.84(63) 127 95.71±0.18(46) 95.89±0.19(85) 95.82±0.28(60) 96.2±0.38(62) 187 92.49±0.46(46) 92.57±0.31(85) 92.91±0.27(60) 92.88±0.56(62) 218 96.03±0.21(46) 96.21±0.27(85) 96.3±0.23(60) 96.46±0.33(62) A) B) c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4 c1 c2 c3 c4