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Sample GSM3611111

Query DataSets for GSM3611111

Status

Public on Nov 15, 2019

Title

151307032_5hmC_Seal

Sample type

SRA

Source name

Plasma

Organism

Homo sapiens

Characteristics

subject status: newly diagnosed patients with DLBCL
race: Blk
Sex: male
age: 49
dlbcl tumor stage: 4
gcb.abc.status: GCB
ldh: 355
ipi.score: 2
deaths=0; alive=1: 1
follow-up time for death (months): 20.6
event: 1; relapse or death
follow-up time for event (months): 20.6
tissue: Plasma
molecule subtype: circulating cell-free DNA (cfDNA)

Extracted molecule

genomic DNA

Extraction protocol

Approximately 2-3 mL of frozen plasma from each subject was processed by centrifuging at 1,350 × g for 12 min twice, then at 13,500 × g for 12 min once, and followed by cfDNA extraction (1-2 ng/sample) using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). Genomic DNA (gDNA) from cfDNA-paired tumor blocks for 7 patients were isolated (30-50 ng/sample) using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany) and fragmented by sonication.
We constructed 5hmC-Seal libraries according to established protocol.22 DNA samples were first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then added to the azide group using Huisgen cycloaddition (“Click”) chemistry. Finally, the 5hmC-containing DNA fragments with biotin tags were captured by avidin beads. The 5hmC-Seal libraries were constructed through PCR amplification and sequenced using the Illumina NextSeq500 platform (PE38) at the University of Chicago Genomics Core Facility. We randomized all samples when preparing the 5hmC-Seal libraries and sequencing. Technical robustness, including reproducibility, of the 5hmC-Seal was demonstrated in our previous report

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Sex: 1; male

Data processing

library strategy: 5hmC-Seal (selective 5hmC chemical labeling)
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 [24] 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 [25](release 19), using featureCounts [26] 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

Submission date

Feb 17, 2019

Last update date

Nov 17, 2019

Contact name

Wei Zhang

E-mail(s)

wei.zhang1@northwestern.edu

Organization name

Northwestern University

Department

Preventive Medicine