Module 9.2 Epigenomic Sequencing Flashcards
Epigenomic sequencing
layers (4)
- DNA methylation
- Histone modifications
- Chromatin accessibility
- Long-range interactions
Chromatin accessibility
- degree to which nuclear micromolecules are able to physically contact chromatin DNA
- chromatin densely arranged within facultative and constitutive heterochromatin
- histones can be replaced by transcription factors at regulatory loci (enhancers, insulators, transcribed gene bodies)
- Transcription factors dynamically compete with histones and other chromatin binding proteins to modulate nucleosome occupancy and promote local access to DNA
- Nucleosome and linker histone occupancy create accessibility spectrum
- chromatin states: closed -> permissive -> open
- Tracing accessibility changes critical to understand epigenetic regulation of gene expression and cellular status
long-range interactions
interactions between regulatory elements across genome
Cytosine converted forms
- Cytosine (C)
- 5-methylcytosine (5-mC)
- 5-hydroxymethylcytosine (5-hmC)
- 5-formylcytosine (5-fC)
- 5-carboxylcytosine (5-caC)
Active demethylation process
- TET family proteins convert 5-mC -> 5-hmC ->5-fC -> 5-caC
- 5-mC: most abundant
- 5-hmC: highly abundant in neurons
- 5-fC & 5-caC: lowest abundance, converted to cytosine via BER enzymes
5-hmC
features
- highly abundant in neurons
- related to activating genes
- plays important role in embryonic development and cell differentiation
- disregulation been shown in tumorogenesis
DNA methylation analysis
bisulfite conversion
features
- unmethylated cytosine converted to uracil via deamination
- methyl group at carbon 5-position (5-mC & 5-hmC) protected against deamination
- denaturation required: sodium bisulfite can only react with cytosine in ssDNA
- PCR: uracils converted to thymines, 5-mC and 5-hmC unchanged
- compared to reference genome
- single nucleotide resolution info about methylation status of DNA segment
post-bisulfite adapter tagging
features
- harsh chemical treatment that damages and nicks DNA
- low library conversion rate, significant loss
- utilize single strand library prep to add sequencing adapters to bisulfite-treated ssDNA
- can salvage fragmented DNA caused by bisulfite treatment to mitigate bisulfite induced loss of sequencing templates
- greatly improved library conversion efficiency
- enabled sequencing with very low sample input eg. single cell
bisulfite conversion
post-bisulfite adapter tagging
methods (3)
- Accel-NGS Methyl-Seq
- TruSeq DNA methylation
- SPLAT Splintered adapter tagging
bisulfite adapter tagging
Accel-NGS Methyl-Seq
process (4)
- Sheared dsDNA undergo bisulfite conversion
- adaptase adds adapters at 3’ end
- single primer extension amplifies 2nd strand with 1st adapter set
- 2nd adapter ligated to both strands at other end
bisulfite adapter tagging
TruSeq DNA Methylation
process
- whole-genome dsDNA undergoes bisulfite conversion
- use random primers to attach adapters to copy of ssDNA
methylation detection
SPLAT
Splinted adapter tagging
- sheared dsDNA undergoes bisulfite conversion
- use adapters with random bases as anchor points for ligation with ssDNA fragment
methylation detection
bisulfite sequencing
benefits and drawbacks
Benefits:
- >99% conversion efficiency of unmodified cytosine to uracil
- original gold standard for mapping 5-mC and 5-hmC
Drawbacks:
- limited read length
- 95% of cytosine in genome converted to T
- 60% AT / 40% GC before treatment -> 75% AT, 20% G & <5% C
- lower mapping efficiency and biased genomic coverage
- makes targeted sequencing more difficult
- unable to distinguish 5mc from 5hmc
methylation detection
Enzymatic Methyl-Seq
(EM-Seq)
features
- converts unmodified C to U
- mild enzymatic reaction with less damage
- longer DNA fragments than bisulfite conversion - easier sequencing and mapping
- enables low sample input (100 picograms and single-cell seq)
- creates low complexity genome
- > 96% protection rate of methylated cytosine, 0.6% false positive rate
- unable to distinguish 5mc from 5hmc
methylation detection
Enzymatic Methyl-Seq
(EM-Seq)
process
- TET2 protein & β glycosyl transferase (βGT) converts all 5-mC and 5-hmC to 5-gmC to protect against deamination
- APOBEC3A deaminates unmethylated cytosine to uracil
- Unmethylated C = T, methylated C = C
methylation detection
TET assisted pyridine borane sequencing
(TAPS)
features
- Directly converts methylated C to T
- novel borane reduction chemistry to convert 5-caC to dihydrouracil (DHU)
- Preserves genomic complexity- only 5% of genomic cytosines are methylated
- higher mapping rate and sequencing quality
- Less destructive chemicals
- Enable lower input DNA
- 96% on 5-mC
- false positive rate ~0.3% on unmethylated cytosine
- not robust system that can be easily reproduced
- unable to distinguish 5mc from 5hmc
methylation detection
TET assisted pyridine borane sequencing
(TAPS)
process
- TET1 oxidation of methylated cytosine to 5-caC
- borane reduction converts 5-caC to DHU
- DHU reads as T after PCR amplification
5-mC / 5-hmC specific detection
methods (6)
5-mC
1. oxBS-Seq
2. TAPSβ
5-hmC
3. ACE-Seq
4. TAB-Seq
5. hmc-CATCH
6. CAPS+
5-mC only detection
TAPSβ
process
- βGT converts 5-hmC to 5-gmC to protect from conversion
- TET1 oxidation converts 5-mC only to 5-caC
- borane reduction converts 5-caC to DHU
- PCR changes DHU to T
Methylation detection
Methylated DNA Immunoprecipitation
(MeDIP)
features
- large scale genome wide affinity based method used to enrich for methylated DNA sequences
- isolate methylated DNA fragments through antibody raised against 5-mC
- DNA fragment size (400-600bp) important for immunoprecipitation efficiency, fragment length bias, and final analysis resolution
- antibody needs more than one 5-MC for efficient binding
Methylated DNA Immunoprecipitation
(MeDIP)
process
- genomic DNA fragmentation (400-600bp) and sequencing adapter ligation
- Denaturation
- ssDNA DNA incubated with monoclonal anti 5-MC antibodies
- Magnetic beads conjugated to anti-mouse IGG used to bind anti 5-MC antibodies (unbound DNA removed in supernatant)
- antibody-bound DNA library molecules eluted and sequenced
Methylated DNA Immunoprecipitation
(MeDIP)
benefits and drawbacks
Benefits
- valuable tool for studying whole genome methylation profiles
- no chemical treatment = minimal damage
- Combining with variant detection enables simultaneous profiling of epigenetics and genetics information in genome
- potentially useful for liquid biopsy
Drawbacks
- provides limited, semi quantitative and low resolution methylation information
- resolution determined by fragment size, usually a few hundred base pairs
- accuracy and reliability heavily rely on specificity and affinity of antibody
- Nonspecific binding or cross reactivity with unmethylated DNA = high noise background or false positive results.
- can’t differentiate 5-mC and 5-hmC
immunoprecipitation methylation detection
hmC-Seal
features
- affinity based method for specifically profiling 5-HMC
- selectively labels 5-HMC for efficient, unbiased, and genome wide profiling
immunoprecipitation methylation detection
hmC-Seal
process
- adapter ligation to create sequencing adapter-incorporated DNA fragments
- fragments treated with βGT enzyme to transfer engineered glusode with azide group to 5-HMC in duplex DNA
- creates modified cytosine with azide group (N3)
- biotin tag installed onto azide group using click chemistry
- 5-hmC-containing DNA fragments with biotin tags efficiently captured from random DNA fragments pool by streptavidin beads
- regular PCR amplification generates sequencing library