Figure 2. Read-depth coverage map of whole-genome bisulphite sequencing dataset (5). (a) The x-axis shows the number of bisulphite sequencing reads at a given cytosine and the y-axis represents number of sites. Most cytosines in all three sequence contexts are covered by <10 reads. For our analysis, only those cytosines were considered for which read depths were between 10 and 50. This proportion, shown in grey, represents 32% of the original data in all three cytosine contexts. (b) Fraction of methyl-cytosines detected at a given sequencing coverage (Read depth). Read depths below 10 lead to an underestimation of methylated CHG and CHH sites, while read depths above 50 tend to be more often associated with methylated cytosines at all three types of sites. and CHH sites within TE sequences are methylated, re- spectively, which is much higher than for genes (26, 4 and 3% for CG, CHG and CHH sites, respectively; File 3 in Supplementary Data). Furthermore, the frequency of methylated sites within the ﬁrst 500 bp beyond genes de- creases 4-fold for CG sites (7%) and remains consistent- ly low for CHG and CHH sites. In contrast, the frequency of methylated CG, CHG and CHH sites is only reduced 2-fold within the ﬁrst 500 bp outside of TE sequences (42, 36 and 16%, respectively). This lower reduction in the frequency of methylated sites outside of TE sequences compared to genes could indicate that our annotation pipeline does not precisely deﬁne TE sequence boundaries or else that DNA methylation can spread from TE sequences into ﬂanking regions (see below). We also note that among CHG sites declared as methylated, a large proportion (>30%) have statistically signiﬁcant discordant methylation levels between the two strands (P < 0.05 in Chi-square goodness of ﬁt test; Supplementary Figure S2). Furthermore, almost all of the latter are devoid of matching siRNAs (data not Nucleic Acids Research, 2011, Vol. 39, No. 16 6923 shown). These ﬁndings indicate that in at least 30% of cases, neither sequence symmetry nor siRNAs play any role in maintaining CHG methylation, which is consistent with this process relying predominantly on a reinforc- ing loop with dimethylation of lysine 9 of histone H3 (2). Methylation status of TE sequences We next determined the DNA methylation status of all individual TE sequences with sufﬁcient information for CG, CHG and CHH sites. Given the repeated nature of TEs and the fact that only sequence reads that map to unique genomic locations with very high conﬁdence are considered (5), cytosine coverage is reduced for TE se- quences (66%) relative to the whole genome (85.6%). Nonetheless, a quarter of cytosines within TE sequences have read depths between 10 and 50, a fraction similar to that for the whole genome (27.2%) and almost identical for CG, CHG and CHH sites. Based on these observa- tions, we only considered the 13 667 TE sequences (43.7% of total) for which information is available for >25% of each of the three distinct types of sites and the 3418 TE sequences (10.9% of total) containing only one (CHH) or two types (CHH and CG or CHG) of sites and still ful- ﬁlling the >25% coverage criterion for these sites. Two main categories of TE sequences are thus excluded from our analysis, those corresponding to recent insertions and for which reads could not be assigned unambiguously because of two or more possible matches in the genome, and those for which technical or other biases lead to <25% coverage for CG, CHG or CHH sites. For each of the 17 085 TE sequences retained for analysis, we determined the methylation status separately for CG, CHG and CHH sites. A sequence was deemed methylated at a given type of site if at least 5% of the sites of this type had reads indicative of methylation, a value that is above the level of non-conversion of unmethylated cytosines [2–4%; (5)]. As illustrated in Figure 3, the fraction of methylated sites within individual TE sequences differs dramatically between CG, CHG and CHH sites. Thus, whereas CG sites are typically all unmethylated or all methylated within a given TE sequence, the fraction of methylated CHG sites varies almost linearly between 0% and 100% (with the exception of a peak at 98–100%) and that of CHH sites rarely exceeds 50%. To simplify the analysis, and because the reason(s) for such wide variations in the frequency of methylated CHG and CHH sites remain to be determined, the methylation status of individual TE sequences was simply summarized as either methylated (M) or un-methylated (U) for each of the three types of sites based on the 5% threshold deﬁned above. This convention leads therefore to eight possible DNA methylation patterns (Table 1), or 10 in the case of TE sequences that are devoid of CG and/or CHG sites (Supplementary Table S1). Although all 18 patterns are observed, few predominate. Thus, among the 13 667 TE sequences with >25% of informative CG, CHG and CHH sites, 58% have an MMM pattern (methylated in all three types of sites) and another 20% have a UUU pattern (unmethylated;