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| Status |
Public on Jul 24, 2023 |
| Title |
Controlserum-A-cfMCC-SEQ |
| Sample type |
SRA |
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| Source name |
Serum
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Serum
cell type: NA
treatment: Control
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Circulating cell-free DNA was extracted from 4 mL human serum or plasma, using the QIAamp Circulating Nucleic Acid kit (Qiagen, Cat. No. 55114) according to the manufacturer’s instructions. Cell-free DNA was quantified via Qubit fluorometer using the dsDNA Assay Qubit fluorometer Kits (ThermoFisher Scientific, Cat. No. Q32853 and Q10212 respectively). As a quality control, fragment size distribution of isolated cfDNA was verified based on analysis using a 2100 Bioanalyzer TapeStation using either a High Sensitivity DNA Assay or DNA 1000 Assay (Agilent Technologies, Santa Cruz, CA, USA) dependent on concentration determined via either Qubit dsDNA BR or HS Assay. Additional purification using Beckman Coulter beads was implemented to remove high-molecular weight DNA reflective of cell-lysis and leukocyte contamination (Agencourt AMPure cat#A63880) (Maggi et al. 2018).
Cell-free DNA from serum was subject to bisulfite conversion using the Zymo EZ DNA Methylation Gold kit (#D5005 Zymo Research, Irvine, CA). Whole Genome Bisulfite Sequencing libraries were generated using the Zymo Research Pico Methyl-Seq Library Prep Kit (#D5455 Zymo Research, Irvine, CA). WGBS libraries were quantified using KAPA Library Quantification Kits for Illumina Platforms (Kapa Biosystems; Wilmington, MA) and then pooled to meet the required 1ug DNA input necessary for targeted enrichment. Hybridization capture was carried out according to the SeqCap Epi Enrichment System protocol (Roche NimbleGen, Inc.; Pleasanton, CA) using xGen Universal Blocker-TS Mix (Integrated DNA Technologies, USA) as the blocking reagent and SeqCap Epi CpGiant probe pools (human) or SeqCap Epi Developer probe pools (mouse samples). MethylC-Capture Sequencing (MCC-seq) libraries were assessed using Qubit Fluorometer (ThermoFisher) and Agilent Bioanalyzer DNA 1000 assays (Agilent Technologies, Inc., USA). MCC-seq libraries with inclusion of 15-20% spike-in PhiX Control v3 library (Illumina; San Diego, CA) were clustered on a Illumina Novaseq 6000 S4 flow cell followed by 150-bp paired-end sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Paired-end FASTQ files were trimmed using TrimGalore (V 0.6.6) with parameters “--paired -q 20 --clip_R1 10 --clip_R2 10 --three_prime_clip_R1 10 --three_prime_clip_R2 10 --retain_unpaired -r1 20 -r2 20” (https://github.com/FelixKrueger/TrimGalore).
Trimmed paired-end FASTQ reads were mapped to the human genome (GRCh37/hg19 build) using Bismark (V 0.22.3) with parameters “--non-directional” (Kreuger et al. 2011), then converted to BAM files using SAMtools (V 1.12) (Li et al., 2009). BAM files were sorted and indexed using Samtools (V1.12).
Reads were stripped from non-CpG nucleotides and converted to BETA and PAT files using wgbstools (V 0.1.0) (Loyfer and Kaplan, 2022).
Assembly: hg19
Supplementary files format and content: BETA, PAT, and index files were generated using wgbstools bam2pat (https://github.com/nloyfer/wgbs_tools) with default parameters
Library strategy: MCC-seq (MethylC-capture sequencing)
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| Submission date |
Aug 26, 2022 |
| Last update date |
Jul 24, 2023 |
| Contact name |
Anton Wellstein |
| E-mail(s) |
anton.wellstein@georgetown.edu
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| Organization name |
Georgetown University
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| Department |
Oncology
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| Lab |
Wellstein Lab
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| Street address |
3970 Reservoir Road, N.W.,
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| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20057 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE200092 |
Circulating cell-free, methylated DNA reveals tissue-specific, cellular damage from radiation treatment [Human Serum MCC-seq] |
| GSE200187 |
Circulating cell-free, methylated DNA reveals tissue-specific, cellular damage from radiation treatment |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6509526_Controlserum_A.beta |
53.8 Mb |
(ftp)(http) |
BETA |
| GSM6509526_Controlserum_A.pat.gz |
20.4 Mb |
(ftp)(http) |
PAT |
| GSM6509526_Controlserum_A.pat.gz.csi |
64.5 Kb |
(ftp)(http) |
CSI |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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