What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?
Each barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.
Are the libraries directional?
Yes, the libraries are directional due to the way our library system is designed and the nature of bisulfite conversion. The forward sequencing reads will correspond to a bisulfite-converted version of either the original top or the original bottom strand (the C-to-T reads) and the reverse sequencing reads will correspond to the complement of the original top or the complement of the original bottom strand (the G-to-A reads). In contrast, a non-directional bisulfite converted library will have all four possible strands in the forward read (original top, original bottom, complement of original top and complement of original bottom).
What analysis software can be used for aligning, methylation calling, and visualization of your bisulfite sequencing data?
The number of analysis strategies and software tools for methylation-based sequencing studies is growing rapidly. The ideal analysis workflow for a given experiment depends on many variables, including the type of experiment and the goals of the study. Currently, Tecan scientists use Bismark for aligning and determining methylation status. This program utilizes the Bowtie aligner (www.bioinformatics.bbsrc.ac.uk/projects/bismark/). The Broad IGV genome browser can be used to visualize the results of Bismark (http://www.broadinstitute.org/igv/). Data analysis recommendations can be found here: https://github.com/tecangenomics/NuMetWG
How can I measure the efficiency of bisulfite conversion?
DNA material that is known to be unmethylated, such as lambda DNA, can be used to measure the efficiency of C-to-U conversion in the bisulfite conversion kit. This control DNA is not included with the Ovation RRBS Methyl-Seq System 1–16.