Bs-ATLAS-seq
Bisulfite-ATLAS sequencing (bs-ATLAS-seq, Figure 1), provides both the location of L1HS insertions and the methylation state of the most distal region of their promoter at single-locus, single-molecule and single-nucleotide resolutions. The amplified region within the L1 5’UTR (210 bp) covers the first 15 CpG dinucleotides - including 7 being considered as critical for L1 regulation - and its methylation level is representative of the broader internal promoter.For the sake of simplicity, we use the term “L1 methylation” to refer to the average DNA methylation of this distal region of the L1 promoter (see Methylation data tab).
A practical protocol for bs-ATLAS-seq is provided here and here.
The internal L1 promoter region (~ 900 bp) is illustrated (top). Transcription start sites for the sense (SP) and antisense (ASP) promoters are represented as broken arrows and overlap with L1 5’UTR. The first seven CpG dinucleotides are considered as critical for L1 regulation. bs-ATLAS-seq interrogates the first 15 CpG sites of the L1 promoter, shown as vertical bars in the magnified view (bottom). The L1-specific primer used to amplify L1 junctions is shown as a green arrow. Genomic DNA is fragmented by sonication and ligated to a single-stranded methylated linker. Linker-ligated DNA is then treated with bisulfite and L1-containing fragments are specifically amplified by suppression PCR. The suppression PCR step is designed to enrich for the L1HS family. Finally, asymmetric paired-end sequencing provides the genomic location as well as the methylation levels of each L1 locus. R1 and R2 refers to reads #1 and #2, respectively. Note that 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are both protected from bisulfite-induced deamination. Thus bs-ATLAS-seq cannot discriminate between these two DNA modifications.