DNA methylation is one of the most studied epigenetic modifications in human cells. Changes in DNA methylation patterns play a critical role in development, differentiation and diseases such as multiple sclerosis, diabetes, schizophrenia, aging, and multiple forms of cancer. Over the past decade, interest in DNA methylation has grown rapidly and expanded across multiple areas of research. Consequently, DNA methylation analysis methods have undergone dramatic changes. Bisulfite-treatment and next-generation sequencing (NGS) have increasingly become the tools of choice to profile DNA methylation levels. Here, we announce a bioinformatics solution for analyzing NGS methylation data: BaseSpace® MethylSeq v1.0 app.
MethylSeq v1.0 app
The core algorithm used in the MethylSeq app is Bismark. Bismark maps bisulfite treated sequencing reads to the genome of interest and performs methylation calls.
The alignment tool used in MethylSeq v1.0 is Bowtie2. Bowtie2 is an ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences.
As with all BaseSpace apps, launching the MethylSeq app on a sample is accomplished with only a few mouse clicks. When the analysis is finished, reports are available via both web browser and PDF. All standard Bismark output files such as BAMs and bedgraph files are also available through the BaseSpace interface. Two demo data sets and screenshots from the MethylSeq app are shown below.
The MethylSeq v1.0 app generates the following output files:
- BAM files, which contain the reads after alignment.
- Bismark Processing Report, which contains a processing report generated by Bismark.
- Cytosine Report (optional), which contains methylation status for every cytosine in the genome, including both strands.
- bedGraph, which contains a cytosine methylation status report for only the cytosines that have sequencing coverage.
The Input form is shown below.
After the user selects the project to save the results to and the sample to analyze, the first option presented is directional or non-directional analysis. The TruSeq DNA Methylation kit is a directional kit, meaning only the original top and bottom strands are sequenced. The reverse complements of the original top/bottom are not. Taking advantage of this directionality significantly improves the performance of the analysis. The remaining options are advanced and described in the application guide. The advanced parameters have been optimized for the TruSeq DNA Methylation kit.
The first two tables in the output report are shown below. They contain information about the number of reads that were analyzed and a table containing the cytosine methylation context. The detailed report can be found by selecting the project in Demo 1 below.
A couple of demo projects are provided below.
An NA18507 dataset generated using the TruSeq DNA Methylation Kit on a NextSeq 550:
BaseSpace project link: https://basespace.illumina.com/s/5qUVcdyaXsSM
Currently, Illumina only supports usage of the MethylSeq app with data generated using the TruSeq DNA Methylation kit (formerly known as EpiGnome). However, this BaseSpace Project demonstrates that the app can potentially work with other bisulfite sequencing preps as well. In this case, Tet-Assisted Bisulfite sequencing (TAB-Seq) and bisulfite sequencing were used to map methylation in the human brain. See the publication for more details: Wen et al. Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain, Genome Biology, 2014.
The data was imported into BaseSpace utilizing the SRA Import app and then processed through the MethylSeq app.
BaseSpace project link: https://basespace.illumina.com/s/gLF8I19cbPG0