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| Status |
Public on Apr 24, 2023 |
| Title |
control pUC19, lambda phage, and T4-phage, 50_mCpG_no_deam |
| Sample type |
SRA |
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| Source name |
control pUC19, lambda phage, and T4-phage
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| Organisms |
Cloning vector pUC19; Escherichia phage T4; Escherichia phage Lambda |
| Characteristics |
sample type: control pUC19, lambda phage, and T4-phage
treatment: 50% CpG methylated
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro derived controls for all other samples.
To compare 5 different sequencing workflows (no deamination, TAPS, TAPS-β, BS-Seq, DM-Seq), 1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, 5mCpG modified Lambda control gDNA, and fully 5hmC-modified T4 phage gDNA) was used for each condition. The 5mCpG modified lambda control gDNA was one of 4 possibilities: fully unmethylated, fully CpG-methylated, fully GpC-methylated, and ~50% CpG-methylated. The ~50% CpG methylated DNA was made by mixing equal amounts of unmethylated and fully CpG-methylated lambda DNA. DNA was end-prepped and ligated with the same protocol as above except for the adapters used, which was different for each method (no deamination, TAPS, and TAPS-β: C adapters, BS-Seq: 5mC adapters, DM-Seq: 5pyC adapters).
BS-Seq was performed using manufacturer instructions (Diagenode). Purified BS-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X) and ultimate characterization using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).
DM-Seq was performed with the optimized protocol. A methylated copy strand was created. 1 μM fully-methylated copy primer was annealed in a total volume of 10 μL in CutSmart Buffer and 1 mM final concentration (individually) of dATP/dGTP/dTTP (Promega) and dmCTP (NEB). 1 μl or 8 units Bst polymerase, large fragment (NEB) was added and incubated for 30 min at 65°C. The 5hmCs were then glucosylated with 40 μM UDP-Glucose and 1 μL or 10 units of T4 Phage β-glucosyltransferase (NEB) for 1 hour at 37°C in a final volume of 20 μL. Incompletely copied or uncopied fragments were degraded with 1 μL or 10 units Mung Bean Nuclease (NEB) for 30 min at 30°C. After SPRI magnetic bead purification (1.2x), libraries were mixed with 0.5 μM MBP-M.MpeI-N374K and 160 μM CxSAM in carboxymethylation buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated overnight at 37°C followed by denaturation for 5 min at 95°C. 1 μL or 0.8 units of Proteinase K (NEB) was subsequently added and incubated at 37°C for 15 min. The samples were purified using SPRI magnetic beads (1.2x) and eluted in 1 mM Tris-Cl, pH 8.0. DNA was then subjected to snap-cooling and A3A deamination in a final volume of 50 μL as previously described before SPRI magnetic beads purification (1.2x). DM-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X). Libraries were then characterized using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).
TAPS and TAPS-β were performed as previously described except for the source of TET enzyme (NEB, EM-Seq Conversion Module). 1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, 5mCpG-modified lambda phage gDNA, and fully 5hmC-modified T4 phage gDNA) was ligated to C-containing Y-shaped adaptors using the same protocol as described above (IDT). For TAPS-β, the ligated DNA was added to a 20 μL reaction containing 1x NEB CutSmart buffer (50 mM Potassium Acetate 20 mM Tris-acetate, 10 mM Magnesium Acetate, and 100 μg/ml BSA), 0.04 nM UDP glucose, and 10 U T4-βGT. The glucosylated DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. This glucosylation step was omitted for TAPS. The purified DNA was then incubated in a 50 μL reaction containing 1x NEB EM-Seq TET buffer (50 mM Tris pH 8.0, 1 mM DTT, 5 mM sodium-L-ascorbate, 20 mM αKG, 2 mM ATP, and 50 mM ammonium iron (II) sulfate hexahydrate) and 16 μg TET2 (NEB). The reaction was then incubated at 37°C for 80 min. Following oxidation, 0.8 U of Proteinase K (NEB) was added, and the mixture was incubated for 30 min at 37°C. The oxidized DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection and input into a second round of TET oxidation. The oxidized DNA was added to a 5 μL reaction containing 600 mM sodium acetate (pH 4.3) and 1 M pyridine borane (Alfa Aesar). The reaction was incubated at 37 °C and 850 r.p.m. in a ThermoMixer (Eppendorf) placed in a chemical fume hood for 16 hrs and purified by Zymo-IC column (Zymo Research) with Oligo Binding Buffer (Zymo Research). The TAPS-β libraries were amplified using indexing primers (NEB) and HiFi Hotstart Uracil+ Ready Mix (KAPA Biosystems) before purification with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. Libraries were ultimately characterized using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
For all workflows, reads were quality and length trimmed with Trim Galore! Reads were aligned with Bismark and deduplicated with Picard. All data was analyzed single-end. DM-Seq reads were filtered if 3 consecutive CpHs were non-converted using Bismark’s existing filter_non_conversion command. Only reads with MAPQ ≥ 30 were analyzed.
Assembly: pUC19, lambda phage ViralProj14204, T4 phage ViralProj14044
Supplementary files format and content: For control DNA samples that were GpC methylated, tab-delimited text files (.txt) include genomic coordinates (chr, start, and end) and deamination (BS-Seq, DM-Seq, TAPS, TAPS-β) analysis (signal [C/(C+T)], sequencing coverage, and strand) of all annotated CpG sites in control genomes. They additionally have the +1 and +2 base identity.
Supplementary files format and content: For control DNA samples where read level information for each control genome was analyzed, aligned files (.bam) are provided, not deduplicated and not filtered. The .bam format makes the number of reads mapping to each genome as well as data reporting percent modification accessible.
Library strategy: no deamination
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| Submission date |
Feb 23, 2023 |
| Last update date |
Apr 25, 2023 |
| Contact name |
Rahul M. Kohli |
| E-mail(s) |
kohlilab@gmail.com
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| Phone |
215-573-7523
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| Organization name |
University of Pennsylvania
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| Department |
Department of Medicine
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| Lab |
Rahul M. Kohli
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| Street address |
3610 Hamilton Walk, 502B Johnson Pavilion
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL33181 |
| Series (2) |
| GSE225971 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution [3] |
| GSE225975 |
Direct enzymatic sequencing of 5-methylcytosine at single-base resolution |
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| Relations |
| BioSample |
SAMN33426300 |
| SRA |
SRX19487503 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7061126_50_mCpG_no_deam.bam |
1.9 Mb |
(ftp)(http) |
BAM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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