title: In silico analysis identifies novel restriction enzyme combinations that expand reduced representation bisulfite sequencing CpG coverage.
abstract: BACKGROUND
  Epigenetics is the study of gene expression changes that are not caused by changes in the deoxyribonucleic acid (DNA) sequence. DNA methylation is an epigenetic mark occurring in C-phosphate-G sites (CpGs) that leads to local or regional gene expression changes. Reduced-representation bisulfite sequencing (RRBS) is a technique that is used to ascertain the DNA methylation of millions of CpGs at single-nucleotide resolution. The genomic coverage of RRBS is given by the restriction enzyme combination used during the library preparation and the throughput capacity of the next-generation sequencer, which is used to read the generated libraries. The four-nucleotide cutters, MspI and TaqαI, are restriction enzymes commonly used in RRBS that, when combined, achieve ~12% genomic coverage. The increase in throughput of next-generation sequencers allows for novel combinations of restriction enzymes that provide higher CpG coverage.
  
  
  RESULTS
  We performed a near-neighbor analysis of the four nucleotide sequences most frequently found within 50 nt of all genomic CpGs. This resulted in the identification of seven methylation-insensitive restriction enzymes (AluI, BfaI, HaeIII, HpyCH4V, MluCI, MseI, and MspI) that shared similar restriction conditions suitable for RRBS library preparation. We report that the use of two or three enzyme combinations increases the theoretical epigenome coverage to almost half of the human genome.
  
  
  CONCLUSIONS
  We provide the enzyme combinations that are more likely to increase the CpG coverage in human, rat, and mouse genomes.
authors.affiliation: The Research Institute of the McGill University Health Centre, Montreal General Hospital, 1650 Cedar Avenue, Room C10-143, Montréal, Québec H3G 1A4, Canada. dan.martinez@mail.mcgill.ca.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Martinez-Arguelles
authors.forename: Sunghoon
authors.initials: S
authors.lastname: Lee
authors.forename: Vassilios
authors.initials: V
authors.lastname: Papadopoulos
mesh_terms: D000818:Animals; D003198:Computer Simulation; D018899:CpG Islands / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D057075:Enzyme Assays; D016678:Genome / Q000235:genetics; D006801:Humans; D051379:Mice; D051381:Rats; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000737:chemistry*
chemical_list: D013447:Sulfites; D004262:DNA Restriction Enzymes; C042345:hydrogen sulfite