human brain genomic dna  (BioChain Institute)

Journal: bioRxiv

Article Title: The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution

doi: 10.1101/2024.12.05.627091

Figure Lengend Snippet: A) Clinker schematic showing the deaminase genomic neighborhood for phage Xp12, the contig encoding mSCD-B5, and phage PaMx74. B ) SDS-PAGE gel showing mSCD-B5 expression and purification. An empty plasmid experiment was included as control. C ) Alphafold rendering of the mSCD-B5 representative dCMP deaminase (green, upper panel) and its closest Foldseek (van ) structural match in the PDB (wheat, lower panel; 7FH4, Chlorovirus PBCV-1 bi-functional dCMP/dCTP deaminase). D ) deamination assay monitored by LC-MS on oligonucleotides containing internal cytosine modifications. Quantification of deamination is shown in the right panel ss: single strand, ds: double strand, mC: 5-methylC, hmC: 5-hydroxymethylC, fC: 5-formylC, caC: 5-carboxyC, gmC: 5-glucosyl-methylC. E ) LC-MS traces (left) and result table (right) of deamination on double strand (-NaOH) or denatured (+NaOH) double strand genomic DNA.

Article Snippet: Human brain genomic DNA from a single donor was purchased from biochain (catalog number: D1234035, LOT: C607510).

Techniques: SDS Page, Expressing, Purification, Plasmid Preparation, Control, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution

doi: 10.1101/2024.12.05.627091

Figure Lengend Snippet: A) Preparation of a 2 fold serial dilution series : A sample containing 4% methylation at dcm sites was obtained by mixing dcm+ genomic DNA (100% methylation at dcm sites) with Δdcm genomic DNA (0% methylation) at a 4:96 molar ratio. Serial 1:2 dilutions were performed by mixing an equimolar amount of Δdcm genomic DNA to the previous dilution, starting from the 4% sample. The serial dilutions generated samples containing methylation levels at 2%, 1%, 0.5%, 0.25%, 0.13%, 0.06% and 0.03% respectively Each dilution level sample was split into two duplicates. The correlation of deamination in 5mC (XP12) on per base resolution between individual samples is displayed as heatmap in the right panel. B ) Percentage of deamination across all 256 NCNNN contexts (where N=A, T, C, or G; log10 scale on y-axis) measured on control genomic DNA samples: XP12 (5mC, left panel), T4gt (5hmC, center-left panel), pUC19 (CpG-context 5mC, center-right panel), and Lambda (canonical C, right panel) for the 10 samples analyzed. Deamination in C pG sites (blue) and C C WGG motifs (orange, where W=A or T) is highlighted to denote CpG methylation in pUC19 and the recognition sites of the Dcm methylase, respectively. C ) Sensitivity curve representing the percentage of deamination across all 256 NCNNN contexts in E. coli DNA mixtures of Δdcm and dcm+ strains. A value of 100% methylation corresponds to DNA from a pure dcm+ strain, while 0% represents DNA from a pure Δdcm strain. Deamination in C C WGG (orange) and CCGGG (red) contexts is highlighted to indicate the Dcm methylase recognition sites and potential “star” activity of the Dcm methylase, respectively.

Article Snippet: Human brain genomic DNA from a single donor was purchased from biochain (catalog number: D1234035, LOT: C607510).

Techniques: Serial Dilution, Methylation, Generated, Control, CpG Methylation Assay, Activity Assay

Journal: bioRxiv

Article Title: The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution

doi: 10.1101/2024.12.05.627091

Figure Lengend Snippet: A) Deamination heatmap in spike-in phage genomes or CG-methylated plasmid pUC19. Unavailable sequence contexts in the pUC19 genome were marked blank. Color scale ranges from 0.0 to 1.0 (100% deamination). Deamination level in CN context in CG-methylated pUC19 plasmid DNA is shown in the right panel. The average deamination percentage in CpG context was 76.1%. B) Percentage of base substitutions in sequencing reads across read positions (x-axis, in base pairs) is shown for two replicates of mSCD-B5-treated libraries (NA12878 genomic DNA, 100 ng input; left two panels) compared to the platinum standard DNA-seq (NA12878). Substitutions were categorized into 12 types, with a specific panel highlighting C-to-T substitutions in CpG contexts to illustrate the magnitude difference compared to other contexts. This breakdown underscores the distinct patterns of C-to-T conversions in CpG (overwhelmingly due to deamination) versus non-CpG regions (from sequencing error or true variants). Only paired-end read 1 were used to avoid the reverse complement of C-to-T (G-to-A) to interfere with baseline substitution C) QC metrics of the fragment size distribution (left panel), GC bias (middle panel) and correlation of the overall-genomic methylation levels obtained with mSCD-B5 (y-axis) and Nanopore (x-axis) at various NCpG contexts (Right panels) for various amount of starting material. D ) Genome-wide methylation level correlation between Nanopore and mSCD-B5 from 0.05 ng to 100 ng starting material. Nanopore base resolution methylation levels were binned into 0-10%, 10-20% until 90-100% (x-axis) and the equivalent methylation from mSCD-B5 were computed and plotted (Y axis) according to the NCpG context (with N=A,T,C or G). E) Methylation levels in CpG island for NA12878 (Left panel) and mixed population of genomic DNA from NA12878 and WT-DKO at ratio 99:1, (Right panel) genomic DNA function of the methylation levels in WT-DKO.

Article Snippet: Human brain genomic DNA from a single donor was purchased from biochain (catalog number: D1234035, LOT: C607510).

Techniques: Methylation, Plasmid Preparation, Sequencing, DNA Sequencing, Genome Wide

Journal: bioRxiv

Article Title: The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution

doi: 10.1101/2024.12.05.627091

Figure Lengend Snippet: A) Deamination heatmap in spike-in phage genomes or CG-methylated plasmid pUC19. Unavailable sequence contexts in the pUC19 genome were marked blank. Color scale ranges from 0.0 to 1.0 (100% deamination) B ) Deamination level in all CN contexts for 5mCpG-methylated pUC19 for BGT treated library (+BGT, Top) and no BGT treatment (-BGT, bottom). The average percentage deamination in CpG context in BGT treated and control samples were 74.7% and 74.8% respectively. C ) Quality control metrics of mSCD-B5-treated libraries derived from human brain genomic DNA. Insert size distribution (in bp, top panels) and GC content bias (bottom panels) for mSCD-B5 + BGT treated libraries (duplicates, left panels) and mSCD-B5 treated libraries (duplicates, right panels). Insert size and GC bias metrics were generated using Picard tools (version 2.0.1)

Article Snippet: Human brain genomic DNA from a single donor was purchased from biochain (catalog number: D1234035, LOT: C607510).

Techniques: Methylation, Plasmid Preparation, Sequencing, Control, Derivative Assay, Generated

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: miR‐146b‐5p expression in 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) treated glioma cell lines (A) as well as methylation of CpG sites located in putative SP1 transcription factor‐binding sites associated with the miR‐146b‐5p locus (B). (A) Relative expression levels of miR‐146b‐5p were determined by real‐time RT‐PCR using let‐7a as reference gene. Expression of the miRNA in the 5‐AZA‐dCR/TSA samples was calculated relative to miRNA expression in the controls (Co). A + T1: cell lines were grown in medium supplemented with 500 nM 5‐AZA‐dCR for 48 h, washed and grown for another 24 h in medium with 500 nM 5‐AZA‐dcr and 1 µM TSA; A + T2: like A + T1, except for a higher 5‐AZA‐dCR concentration (1 µM). Asterisks indicate significant difference when compared with the respective non‐treated controls (*P < 0.05; **P < 0.01; paired t‐test). Bars represent the mean, and error bars represent the standard deviation of three independent experiments, except for U138MG A + T2 with two independent experiments. (B) Methylation analysis of the seven CpG sites located from nt 104,195,358 to nt 104,195,483 on chr10 (UCSC genome browser, GRCh37/hg19 assembly) was performed by pyrosequencing of two PCR products generated from sodium bisulfite‐modified DNA. The three CpG sites located within each of the three putative SP1‐binding sequences are labeled by hachures. The methylation status at each CpG site was gray scale‐coded as follows: white square, 0–25% methylated alleles; light gray square, 26–50% methylated alleles; gray square, 51–75% methylated alleles; black square, 76–100% methylated alleles. methCo, methylation positive control (CpGenome™ Universal Methylated DNA, Merck Millipore, Darmstadt, Germany); AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue; Meth% (mean), mean of the percentages of methylated allele derived from pyrosequencing at the seven investigated CpG sites. IDH mut: gray square, IDH1 or IDH2 mutation detected; white square, no IDH1 or IDH2 mutation detected. TERT mut: gray square, TERT promoter mutation detected; white square, no TERT promoter mutation detected. Note a significant difference in the mean percentage of methylated alleles between primary glioblastoma (mean: 5% methylated alleles) and secondary glioblastoma (mean: 30% methylated alleles; P < 0.01; Student's t‐test, two‐sided).

Article Snippet: Three different commercially available genomic DNA samples (BioChain) from human brain tissue served as reference for DNA methylation analyses.

Techniques: Expressing, Methylation, Binding Assay, Quantitative RT-PCR, Concentration Assay, Standard Deviation, Generated, Modification, Labeling, Positive Control, Derivative Assay, Mutagenesis

Journal: Brain Pathology

Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas

doi: 10.1111/bpa.12294

Figure Lengend Snippet: miR‐346 expression in 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) treated glioma cell lines as well as methylation of the miR‐346‐associated CpG island in astrocytic gliomas. (A) Relative expression levels of miR‐346 were determined by real‐time RT‐PCR using let‐7a as reference. Expression of miR‐346 in the A + T1 and A + T2 samples was calculated relative to the expression in the controls (Co). A + T1: Cell lines were grown in medium supplemented with 500 nM 5‐AZA‐dCR for 48 h, washed and grown for another 24 h in medium with 500 nM 5‐AZA‐dCR and 1 µM TSA; A + T2: like A + T1, except for a higher 5‐AZA‐dCR concentration (1 µM). Asterisks indicate significant difference as compared with the respective non‐treated (‐) controls (***P < 0.001; paired t‐test). Bars represent the mean, and error bars represent the standard deviation of three independent experiments, except for U138MG A + T2 with two independent experiments. (B) Methylation analysis of the 9 CpG sites of the CpG: 47 island (chr10; UCSC genome browser, GRCh37/hg19 assembly) located between nucleotides 88,023,052 and 88,023,105 in intron 1 of GRID1 was performed by pyrosequencing of a PCR product generated from sodium bisulfite‐modified DNA. The methylation status at each CpG site was gray scale‐coded as followed: white square, 0–25% methylated alleles; light gray square, 26–50% methylated alleles; gray square, 51–75% methylated alleles; black square, 76–100% methylated alleles; methCo, methylation positive control (CpGenome™ Universal Methylated DNA, Merck Millipore, Darmstadt, Germany); AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissues. Meth% (mean), mean of the percentages of methylated allele derived from pyrosequencing at the nine investigated CpG sites. IDH mut: gray square, IDH1 or IDH2 mutation detected; white square, no IDH1 or IDH2 mutation detected. Note a significant difference in the mean percentage of methylated alleles between primary glioblastoma (mean: 48% methylated alleles) and secondary glioblastoma (mean: 27% methylated alleles; P < 0.01; Student's t‐test, two‐sided).

Article Snippet: Three different commercially available genomic DNA samples (BioChain) from human brain tissue served as reference for DNA methylation analyses.

Techniques: Expressing, Methylation, Quantitative RT-PCR, Concentration Assay, Standard Deviation, Generated, Modification, Positive Control, Derivative Assay, Mutagenesis