Paper - Review
10.1038/nprot.2010.190
DOI: 10.1038/nprot.2010.190
Abstraction
Genome-wide mapping of 5-methylcytosine
→ interest to many fields of (biology & medicine)
⭐ RRBS (Reduced Representation Bisulfite sequencing)
→ a bisulfite-base protocol (→ which enriches CG-rich parts of the genome)
∴ RRBS reduce ↓ the amount (← of sequencing required) while capturing the majority of (promoters & other relevant genomic regions)
⭐ RRBS
→ provides single-nucleotide resolution
→ (is highly ↑ sensitive) & (provides quantitative DNA methylation measurements)
→ enable (any standard molecular biology lab) to generate RRBS libraries of high quality
RRBS
← with extremely low input requirements (10-300 ng)
→ the applicability of the protocol to FFPE samples
Introduction
DNA methylation (← in mammal)
→ within the context of the symmetric CpG dinucleotide
→ its distribution is uneven (← with the majority of the genome containing very few ↓ CpGs)
CpG islands
→ are often un-methylated (← across cell types)
Genome-scale studies
→ show that (the effect is largely dependent on the genomic location)
DNA methylation
→ has a repressive effect (← in promoter & enhancer regions)
→ be enriched (← in gene bodies of actively transcribed gene)
Development of the protocol
(Uneven distribution of CpG dinucleotides) & (Short read-length of NGS approaches)
→ make whole-genome shotgun bisulfide sequencing (too costly & inefficient) → for comparing (hundreds of mammalian samples)
∴ Overcome → Designed a strategy to generate small fragments (← that compose only ~1% of the genome)
∴ each (end-sequencing read) covers (informative CpG methylation measurement)
Applications of the method
RRBS
→ the coverage of important genomic features → while (only requiring) a modest amount of sequencing
→ high sensitivity
→ allow to use FFPE samples
∴ RRBS is highly suited to study (the role of DNA methylation)
Comparisons with other methods
3 categories (← for DNA methylation mapping)
1) Bisulfite-based approaches (← including RRBS & Illumina arrays)
2) Digestion with methylation-sensitive restriction enzymes (← including HELP & CHARM)
3) Capture of methylated DNA fragments (← including MeDIP & MBD capture)
The application of each
← depends on (availability to the user) & (requirements)
Limitation of the approach
RRBS
→ enriches for genomic regions (← which contain CpG dinucleotides)
→ captures (the majority of CpG islands & promoters)
→ also provides (coverage of many other genomic regions)
→ provides an informative sampling (← of the genome)
→ The coverage can be varied to some extent (← by the size-selection & choice of enzymes)
Overview of key steps in the RRBS Protocol
⭐ Major advantage of RRBS
→ its reproducibility over (time & different sample types)
RRBS
→ relies on a binary (← rather than a quantitative read-out)
→ decouples (the DNA methylation measurement)
Genomic DNA purification
Purity (← of the genomic DNA)
→ is critical (← to generate an optimal RRBS library)
Cellular proteins
← especially the DNA binding proteins
→ interfere with downstream enzymatic reactions
Excessive amounts of (cellular RNA)
→ have adverse effects
e.g. effecting DNA quantification
Restriction endonuclease digestion
Many restriction endonucleases can be used → to generate (reduced representations)
Other enzymes can be applied → to generate reproducible (genome fractionation)
Every MspI-digested fragment ← contains (2 terminal CpGs)
MspI digest (← in silico) ← of vertebrate genomes
→ allows prediction of (the number of expected DNA fragments) & (the size distribution)
Filling in a A-tailing of MspI-digested DNA fragments
A single reaction is carried out
→ to fill-in the 3'-terminal recessive ends (← in the MspI-digested fragments)
→ to add an extra A (← to both plus & minus strands)
Methylated-adapter ligation
The methylated adapter oligos
← cytosine is replaced by 5-methylcytosine (← to maintain integrity)
→ are (annealed & result) in fork structures
→ dsDNA would on a Criterion precast poly-acylamide TBE gel
A-tailed DNA fragments
→ must be ligated to (the Illumina standard) & (paired-end adapters) on both ends
The use of (the standard) & (paired-end adapters)
← depends on the goal of the study
Size selection of the adapter-attached DNA fragments
In silico digest of vertebrate genomes
→ a fraction of 40-220 bp of DNA fragments (← which contains representative coverage)
→ enrichment of (most promoter sequences) & (CpG island regions)
❗To isolate (this ↑ fraction) (← of the genome) from the adapter-ligated DNA
→ Lengths of (50 & 60 bp) should be added to the desired size range of (the standard) & (the paired end) adapter-attached DNA
∵ the specific structures of (the standard adapters) & (paired-end adapters)