The anchor is a crucial part of any seaworthy ship of exploration, and after setting sail from your local CpG island, you’ll be glad you stowed anchor-based bisulfite sequencing (or ABBS) to keep your DNA methylation voyages firmly anchored to new and exciting regions of the methylome.
Anchor-based bisulfite sequencing, which comes to you from a team of seafaring Californians led by Benjamin Delatte from Active Motif (Carlsbad, California), employs anchored oligonucleotides to target sequences bearing DNA methylation after bisulfite conversion. This novel protocol provides base resolution results in a single day, using ten times fewer reads than whole-genome bisulfite sequencing (WGBS)!
Let’s hear from Chapin and colleagues about their voyage through the methylome with anchor-based bisulfite sequencing:
- Genomic DNA treatment with sodium bisulfite at high temperatures converts unmodified cytosines to uridines (leaving methylated cytosines unaffected), induces DNA fragmentation, and denaturation into single-stranded DNA
- Subsequent sonication generates 200-300 nucleotide fragments
- After this preparation, the addition of a primer comprising five random nucleotides and a 3′ anchor of PPG (a pyrazolo[3,4-d]pyrimidine analog that stabilizes PPG:C base-pairing compared to canonical guanosines) targets methylated regions
- The anchored primer undergoes perfect 3′ hybridization at cytosines remaining after bisulfite treatment (i.e., methylated cytosines), allowing later elongation with Klenow (exo minus) polymerase
- While the anchor nucleotide specifically targets methylated sites, the sequencing accurately measures surrounding methylated and unmethylated cytosines
- Applying ABBS in E. coli demonstrates that anchored primers efficiently target expected DNA methylation sites and redirects sequencing power to methylated regions
- Comparative analyses in human cancer cells highlight the correlation of ABBS-measured methylation levels with WGBS
- ABBS also selectively targets the methylated genome to a similar extent as methylated DNA immunoprecipitation-seq but provides a broader view of the methylation landscape than reduced representation bisulfite sequencing
- ABBS reduces the sequencing coverage needed for genome-wide DNA methylation mapping up to tenfold compared to WGBS
ABBS finds port after its maiden voyage, where this exciting new technique proved itself as a ship-shape and cost-effective means to explore the methylome that may keep more commonly employed DNA methylation profiling techniques all at sea!
Senoir author Benjamin Delatte shares that “because of its low sequencing cost, ABBS positions itself as a great alternative for analyses of a large amount of samples and meta-epigenomic studies.”
Set sail from your CpG island and anchor yourself to new regions of the genome with anchor-based bisulfite sequencing in Communications Biology, June 2022.