Thursday 23 May 2019
Friday 17 May 2019
Wednesday 15 May 2019
Differentially Methylated Loci vs Differentially Methylated Regions
Cited from the paper "BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions"
"""
However, various authors have noted that methylation levels are strongly correlated across the genome [24, 25]. Furthermore, functionally relevant findings are generally associated with genomic regions rather than single CpGs, either CpG islands [26], CpG island shores [27], genomic blocks [1], or generic 2 kb regions [3].
"""
"""
However, various authors have noted that methylation levels are strongly correlated across the genome [24, 25]. Furthermore, functionally relevant findings are generally associated with genomic regions rather than single CpGs, either CpG islands [26], CpG island shores [27], genomic blocks [1], or generic 2 kb regions [3].
"""
Tuesday 14 May 2019
MethylC-seq or BS-seq
Cited from the paper "MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing"
"""
MethylC-seq library preparation protocol overview. gDNA (i) is fragmented to ~200 bp by sonication (ii). DNA fragments containing damaged or incompatible 5′- and/or 3′-protruding ends are converted to 5′-phosphorylated, blunt-ended DNA (iii). Blunt-ended DNA fragments are converted to DNA with 3′-dAMP overhangs (iv). Methylated Y-shaped adapters are ligated to the dA-tailed DNA fragments (v). All cytosines in the adapters must be methylated to allow for primer binding and amplification after bisulfite conversion. Adapter-ligated DNA fragments are denatured, and unmethylated cytosine is converted to uracil during sodium bisulfite treatment (vi). Bisulfite-treated DNA fragments remain single-stranded as they are no longer complementary. Low-cycle PCR amplification is performed with a polymerase that can tolerate uracil residues (vii). The final library fragments contain thymines and cytosines in place of the original unmethylated cytosines and methylated cytosines, respectively (viii).
"""
"""
MethylC-seq library preparation protocol overview. gDNA (i) is fragmented to ~200 bp by sonication (ii). DNA fragments containing damaged or incompatible 5′- and/or 3′-protruding ends are converted to 5′-phosphorylated, blunt-ended DNA (iii). Blunt-ended DNA fragments are converted to DNA with 3′-dAMP overhangs (iv). Methylated Y-shaped adapters are ligated to the dA-tailed DNA fragments (v). All cytosines in the adapters must be methylated to allow for primer binding and amplification after bisulfite conversion. Adapter-ligated DNA fragments are denatured, and unmethylated cytosine is converted to uracil during sodium bisulfite treatment (vi). Bisulfite-treated DNA fragments remain single-stranded as they are no longer complementary. Low-cycle PCR amplification is performed with a polymerase that can tolerate uracil residues (vii). The final library fragments contain thymines and cytosines in place of the original unmethylated cytosines and methylated cytosines, respectively (viii).
"""
Comprehensive high‑throughput arrays for relative methylation (CHARM)
Cited from the paper "Comprehensive High-Throughput Arrays for Relative Methylation (CHARM)"
"""
Overview of McrBC-based fractionation (Lippman et al., 2005; Ordway et al., 2006) coupled with CHARM analysis (Irizarry et al., 2008). Genomic DNA is sheared to 1.5 to 3.0 kb and divided into two equal parts. The first is digested with McrBC, a methyl-cytosine insensitive enzyme that recognizes PumC(N40–3000)mCPu, and the second is untreated. Both fractions are then resolved side by side on a 1% agarose gel, and fragments between 1.65 kb and 3.0 kb are excised and purified. Next, the untreated fraction, representing total input DNA, is labeled with cyanine-3 (Cy3) and the McrBC-treated fraction, representing unmethylated DNA, is labeled with cyanine-5 (Cy5) followed by cohybridization to a CHARM microarray. Sequences that are methylated will be present in the input fraction (Cy3) and depleted in the methyl-depleted fraction (Cy5). For each probe on the array, a log ratio of the Cy3 to Cy5 intensity is calculated and represents the methylation level (M-value) at each locus, with larger M-values representing more methylation and smaller M-values representing less methylation.
"""
Cited from the paper "Profiling genome-wide DNA methylation"
"""
McrBC, an enzyme that digests methylated DNA, to fractionate DNA and subsequently utilizes array hybridization.
"""
"""
Overview of McrBC-based fractionation (Lippman et al., 2005; Ordway et al., 2006) coupled with CHARM analysis (Irizarry et al., 2008). Genomic DNA is sheared to 1.5 to 3.0 kb and divided into two equal parts. The first is digested with McrBC, a methyl-cytosine insensitive enzyme that recognizes PumC(N40–3000)mCPu, and the second is untreated. Both fractions are then resolved side by side on a 1% agarose gel, and fragments between 1.65 kb and 3.0 kb are excised and purified. Next, the untreated fraction, representing total input DNA, is labeled with cyanine-3 (Cy3) and the McrBC-treated fraction, representing unmethylated DNA, is labeled with cyanine-5 (Cy5) followed by cohybridization to a CHARM microarray. Sequences that are methylated will be present in the input fraction (Cy3) and depleted in the methyl-depleted fraction (Cy5). For each probe on the array, a log ratio of the Cy3 to Cy5 intensity is calculated and represents the methylation level (M-value) at each locus, with larger M-values representing more methylation and smaller M-values representing less methylation.
"""
Cited from the paper "Profiling genome-wide DNA methylation"
"""
McrBC, an enzyme that digests methylated DNA, to fractionate DNA and subsequently utilizes array hybridization.
"""
Isoschizomers and Neoschizomers
Cited from Wiki
"""
Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
"""
"""
An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer . Neoschizomers are a specific type (subset) of isoschizomer. For example, SmaI (CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of each other.
"""
"""
Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.
"""
"""
An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer . Neoschizomers are a specific type (subset) of isoschizomer. For example, SmaI (CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of each other.
"""
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