6924 Nucleic Acids Research, 2011, Vol. 39, No. 16 Figure 3. Frequency of methylated CG, CHG and CHH sites in TE sequences. (a) Boxplots showing frequency distribution of methyl- cytosines for TE sequences methylated for at least one type of site. Most of these TE sequences have almost all of their CG sites and a majority of their CHG sites methylated. (b) Frequency distribution of all TE sequences in relation to percentage of methylated sites, for each of the three types of sites. Table 1). Moreover, the next most abundant pattern is UUM (6%), which is characterized by a particularly low median level of methylation for methylated CHH sites and a low-median frequency of such sites (1% and 8%, respectively, compared to 6% and 36% for the MMM pattern). Thus, whereas a majority of TE sequences for which information is available for CG, CHG and CHH sites are densely methylated, a large proportion (26%) are not signiﬁcantly methylated at any of the three types of sites. Another 10% of TE sequences are methylated mainly at CG sites (MUM, MMU and MUU patterns) and have low median levels of methylation and frequency of methylated CG sites (12–40% and 38–75%, respect- ively, compared to 82% and 100% for the MMM pattern). These TE sequences therefore have methylation patterns resembling those of methylated genes. Finally, a small proportion of TE sequences exhibit non-CG methylation only (UMU and UMM patterns, 5% of total). Methylation data obtained by immunoprecipitation of DNA with an anti-methylcytosine antibody followed by hybridization to a high-density genome-tiling microarray [MeDIP chip; (22)] were used to validate the pertinence of the main and most contrasted patterns MMM and UUU+UUM. Out of the 382 178 probes on the array, 14 482 were extracted that covered, with little risk of cross-hybridization (see ‘Materials and Methods’ section), 4272 MMM and 1433 UUU + UUM TE se- quences. Overall, 75 and 98% of probes corresponding to MMM and UUU + UUM patterns were declared as methylated and unmethylated, respectively, validating our classiﬁcation and indicating a higher sensitivity of bisulphite sequencing over MeDIP in detecting methylated DNA, as previously reported (26). Among the 3418 TE sequences devoid of CG and/or CHG sites and with >25% of informative sites of the other type(s), 42% have patterns ( U, -UU and U-U) clearly indicative of no, or very low, methylation (Supplementary Table S1). The -UM and U-M patterns (8% of total) also indicate very low methylation as they Table 1. Methylation patterns for 13367 TE sequences with CG, CHG and CHH sites MMM UUU UUM MUM MUU UMU MMU UMM No. TEs 7983 2687 Percentage TEs 0.58 0.20 Nb TEs in Heterochromatin 5802 591 Nb TEs in Euchromatin 2181 2096 Average size (bp) 887 443 Average size in Heterochromatin 929 398 Average size in Euchromatin 777 456 Average distance* 0.24 0.27 Median Frequency of methylated sites CG 1.00 0.00 CHG 0.90 0.00 CHH 0.36 0.00 Median level of methylation (methylated reads/total reads) CG 0.82 0.00 CHG 0.28 0.00 CHH 0.06 0.00 *Jukes–Cantor distance from reference sequence. 768 0.06 205 563 396 332 419 0.27 0.00 0.00 0.08 0.00 0.00 0.01 554 0.04 279 275 443 453 433 0.26 0.75 0.00 0.12 0.40 0.00 0.01 469 0.03 175 294 591 589 593 0.26 0.38 0.00 0.02 0.12 0.00 0.00 453 0.03 113 340 635 583 652 0.27 0.00 0.17 0.02 0.00 0.01 0.00 449 0.03 210 239 679 612 738 0.26 0.60 0.29 0.03 0.37 0.03 0.00 304 0.02 82 222 488 440 506 0.27 0.00 0.25 0.10 0.00 0.02 0.01