Abstract
Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.
Highlights
The MCSeEd technique was used to monitor DNA methylation changes induced by drought stress in maize leaves
We have developed a reduced-representation, reference-free approach for characterizing whole genome methylation patterns across different methylation contexts
Alternative approaches aimed at reducing the sequencing demands are based on genomic digestion with either methylation-sensitive or methylation-insensitive endonucleases, before direct next-generation sequencing (NGS) (e.g., EpiRADseq[30], MRE-seq29), or bisulfite treatments and NGS (e.g., RRBS27, methylation-sensitive restriction enzyme bisulfite sequencing [MREBS]41)
Summary
Since one of our goals was to develop a high-throughput technique that can be applied to species without a reference genome, the MCSeEd bioinformatic pipeline was tested for mapping of filtered reads to a pseudo-reference genome autogenerated by a pipeline, hereafter indicated as the genome independent strategy
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
