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Status |
Public on Jan 31, 2020 |
Title |
SEQ811 |
Sample type |
SRA |
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Source name |
Plasma
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Organism |
Homo sapiens |
Characteristics |
tissue: Plasma disease diagnosis: Type 2 Diabetes population: Chinese sample: F25 batch: 1 t2dm_complications: 3 t2dm_nephropathy: 0 age: 62 binary for gender: 2 athero: 1 t2dm_comp: Yes age_bin: 1 multiple_single: Multiple
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Extracted molecule |
genomic DNA |
Extraction protocol |
CfDNA samples from T2D patients were prepared from peripheral blood with EDTA anticoagulation. Briefly, 4 mL of peripheral blood was collected from each subject, and plasma was separated within 4 h by centrifuging twice, one at 1 350× g for 12 min, and another at 13 500× g for 5 min. The processed plasma samples were stored at –80 °C immediately. CfDNA was extracted using the Circulating Nucleic Acid Kit (Qiagen, Germany) according to the manufacturer’s instructions. On average, 1-2 ng of cfDNA was obtained from each cfDNA sample. The quality of cfDNA was checked using the standard molecular biology approach. Each cfDNA sample was first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase (β-GT) was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then installed onto the azide group using Huisgen cycloaddition (click) chemistry, followed by capturing of 5hmC-containing DNA fragments using avidin beads. The 5hmC-Seal library for each cfDNA sample was then constructed through PCR amplification, followed by paired-end sequencing (PE38) at Shanghai Epican Genetech, Co. Ltd. (Shanghai, China), using the Illumina NextSeq500 platform according to Illumina’s protocol. We randomized cfDNA samples when constructing the 5hmC-Seal libraries and sequencing.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NextSeq 500 |
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Description |
F25 cfDNA processed data file: 5hmC_Count_Matrix.csv
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Data processing |
Library strategy: 5hmC-Seal Raw sequencing reads were trimmed for adaptor sequences using Trimmomatic. Low quality bases were also trimmed to a minimum length of 30 bp, followed by alignment to the current human genome reference (hg19) using Bowtie2 with the end-to-end alignment mode. Read pairs were concordantly aligned with fragment length ≤ 500 bp and with average ≤ 1 ambiguous base and up to four mismatched bases per 100 bp length. Alignments with Mapping Quality Score ≥ 10 were counted for gene bodies, according to the gene start and gene end annotations by the GENCODE Project (release 19), using featureCounts without strand information. Peaks were called using PeaksFind version 2.2 with the following setting: ChIP threshold (0.2), Enrichment Fold (2.5), Rescue Fold (3). The 5hmC-Seal libraries were sequenced to produce a median number of ~25 million reads in cfDNA samples, and a median number of ~13.5 million unique reads (~55% of total reads) were mapped to the ~22,000 gene body regions. Genome_build: hg19 (GRCh37) Supplementary_files_format_and_content: 5hmC_Count_Matrix.csv: Comma-separated text file
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Submission date |
Jan 30, 2019 |
Last update date |
Jan 31, 2020 |
Contact name |
Wei Zhang |
E-mail(s) |
wei.zhang1@northwestern.edu
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Organization name |
Northwestern University
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Department |
Preventive Medicine
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Street address |
680 N Lake Shore Drive, Suite 1400
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City |
Chicago |
State/province |
IL |
ZIP/Postal code |
60611 |
Country |
USA |
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Platform ID |
GPL18573 |
Series (1) |
GSE125929 |
5-Hydroxymethylcytosines in Circulating Cell-free DNA Reveal Vascular Complications of Type 2 Diabetes |
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Relations |
BioSample |
SAMN10849433 |
SRA |
SRX5313348 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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