Fig 1.
The analysis workflow of TABSAT is shown.
The first step conducts quality assessment and generation of a report. Next, sequences are mapped to a reference genome and the methylation information is extracted. Based on several quality statistics and thresholds the generated results are filtered and aggregated into a final output table. In the next step, reads covering all CpGs in a target region are used to calculate methylation-pattern statistics, which are subsequently compared between samples. The last step creates the final output table, graphical representations of the results, and reports basic statistics generated during the analysis workflow.
Fig 2.
The Platomics input page of TABSAT is displayed.
After setting the correct parameters, a new analysis is started by clicking on the “Start Job” button. Running and finished analyses are displayed in the jobs panel and can be selected by clicking on the corresponding job.
Fig 3.
The result of a TABSAT run using the Platomics platform is shown.
The output table of a run is presented in the upper part of the large panel containing the sequencing results for each CpG site of all samples. The lower panel shows the graphical representation of the methylation results. Additional information, such as mapping statistics and quality control information, about each sample can be accessed by clicking on the corresponding links.
Fig 4.
This image illustrates the determination and comparison of methylation patterns.
The top line shows an example target region where the CpG coordinates are highlighted. Displayed below are several reads mapping to this target with different methylation patterns. From these reads methylation-patterns are extracted, ranked according to their frequency, and the patterns are compared between samples.
Fig 5.
Displayed is an example of the two automatically generated lollipop diagrams containing the CpGs of the target region: a) all CpGs equally spaced across the target; b) all CpGs spaced according to their true chromosomal coordinate.
Table 1.
Output comparison using 16 datasets between the standard version and the Ion Torrent optimized version of Bismark.
Parameters standard version: -bowtie2—non_directional; parameters Ion Torrent version: -tmap—non_directional.
Fig 6.
An example target region with marked CpG positions and forward/reverse primers is shown.
The 450k target is depicted below as well as the CpGs measured using targeted sequencing. Grey color intensity is correlated to the methylation signal (450k) and percent methylation (sequencing).
Fig 7.
Presented is the correlation result between the Illumina 450k methylation chip and targeted bisulfite sequencing data: a) Barplot of the correlation for each sample; b) Scatterplot of 450k (x-axis) versus targeted bisulfite data (y-axis).
Table 2.
Details of the used amplicons: position on the human reference genome hg19, 450k methylation site ID, length of the target, number of CpGs, melting temperature, GC percentage, and average number of mapped reads (per sample).