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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 (6mA). 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, 6mA). 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.

Number of reads generated upon sequencing of eight C. boidinii strains. (DOCX 15 kb)

Number of genomic bases included in transposable elements, simple repeats and low complexity regions of eight C. boidinii strains. (DOCX 14 kb)

Number of predicted genes showing high homology (e-value

Number of predicted genes showing high homology (e-value

Alignment statistics for the Blast search of two D1D2 ribosomal portions (isolated and sequenced from one high GC and one low GC content strain) in the eight C. boidinii strains. (DOCX 15 kb)

Number of genomic bases included in transposable elements, simple repeats and low complexity regions of eight C. boidinii strains. (DOCX 14 kb)

Gene Ontology enrichment analysis of the Bur0 accession transcripts that are enclosed in gained regions (P = process and F = function). (DOC 21 kb)

Sequence read archive (SRA) experiments for A. thaliana accessions Bur0 and Tsu1 available at http://www.ncbi.nlm.nih.gov/sra. (DOC 209 kb)

Pileup analyser parameters to detect the simulated polymorphisms in the A. thaliana genome with different reference coverage values. (DOC 207 kb)

G|C bias in the Bur0 and Tsu1 Illumina NGS datasets. (DOC 21 kb)

Gene Ontology enrichment analysis of the Bur0 accession transcripts that are enclosed in lost regions, including copy number variation and zero coverage reference genome portions (P = process, F = function and C = cellular component). (DOC 215 kb)

Comparison of the intron sizes between Dictyostelium discoideum and Dictyostelium purpureum. All the introns annotated in these two genomes were compared, including 15,510 in Dictyostelium discoideum and 18,412 in Dictyostelium purpureum; Table S3. The abundance of repetitive sequences and introns in Dictyostelium discoideum and Dictyostelium purpureum. Table S4. Exonic sequences flanking lost introns have higher GC contents than those flanking conserved introns in most previously studied organisms; Table S5. At discordant intron positions, the relative GC content of exonic sequences flanking lost introns compared with the exonic sequences flanking extant introns of the sister species; Table S6. The GC content of exonic sequences flanking extant introns at discordant intron positions compared with those flanking conserved intron sites of the same species; Table S7. Version numbers and source databases of the plant and animal genomes used in this study. (DOC 540Â kb)

Results of gene ontology enrichment analysis. (DOC 203Â kb)

Comparison of the intron sizes between Dictyostelium discoideum and Dictyostelium purpureum. All the introns annotated in these two genomes were compared, including 15,510 in Dictyostelium discoideum and 18,412 in Dictyostelium purpureum; Table S3. The abundance of repetitive sequences and introns in Dictyostelium discoideum and Dictyostelium purpureum. Table S4. Exonic sequences flanking lost introns have higher GC contents than those flanking conserved introns in most previously studied organisms; Table S5. At discordant intron positions, the relative GC content of exonic sequences flanking lost introns compared with the exonic sequences flanking extant introns of the sister species; Table S6. The GC content of exonic sequences flanking extant introns at discordant intron positions compared with those flanking conserved intron sites of the same species; Table S7. Version numbers and source databases of the plant and animal genomes used in this study. (DOC 540Â kb)

Presence and absence of introns at the discordant positions among the five genomes studied. (DOC 1161Â kb)