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| Status |
Public on Jun 22, 2020 |
| Title |
Subject 3 - low DNA input (<300ng) |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Peripheral blood mononuclear cells
dna input: Low DNA input
dna input quantity: <300ng
subject: 3
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from de-identified PBMC collected from four individuals. Genomic DNA quality was determined by estimating the A260/A280 and A260/A230 ratios by spectrophotometry and concentration by fluorometry. DNA integrity and fragment size were confirmed using a microfluidic chip run on an Agilent Bioanalyzer. To assess the reproducibility of MC-seq by DNA quantity, DNA samples from each participant were profiled in triplicate times with high (>1000ng), medium (300-1000ng) and low (150-300ng) DNA input. In total, 12 DNA samples were measured by MC-seq.
Indexed paired-end whole genome sequencing libraries were prepared using the SureSelect XT Methyl-Seq kit (Agilent, part#G9651B). Genomic DNA was sheared to a fragment length of 150-200 bp using focused acoustic energy delivered by the Covaris E220 system (Covaris, part#500003). Fragmented sample size distribution was determined using the Caliper LabChip GX system (PerkinElmer, Part#122000). Fragmented DNA ends were repaired with T4 DNA Polymerase and Polynucleotide Kinase and “A” base was added using Klenow fragment in a single reaction followed by AMPure XP bead-based purification (Beckman Coulter, part#A63882). The methylated adapters were ligated using T4 DNA ligase followed by AMPure XP bead purification. Quality and quantity of adapter-ligated DNA were assessed using the Caliper LabChip GX system. Samples yielding >350 ng were enriched for targeted methylation sites by using the custom SureSelect Methyl-Seq Capture Library. Hybridization was performed at 65°C for 16 hours using a C1000 Thermal Cycler (BIO-RAD, part# 1851197). Once the enrichment was completed the samples were mixed with streptavidin-coated beads (Thermo Fisher Scientific, part#65602) and washed with a series of buffers to remove non-specific bound DNA fragments. DNA fragments were eluted from beads with 0.1 M NaOH. Unmethylated C residues of enriched DNA were modified by bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, part#D5005). The SureSelect enriched, bisulfite-converted libraries were PCR amplified using custom-made indexed primers (IDT, Coralville, Iowa). Dual-indexed libraries were quantified by quantitative polymerase chain reaction (qPCR) using the Library Quantification Kit (KAPA Biosystems, Part#KK4854) and inserts size distribution was assessed using the Caliper LabChip GX system. Samples with a yield of ≥2 ng/ul were proceeded to sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Methylation capture bisulfite sequencing
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| Data processing |
Library strategy: MC-seq
Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Sample de-multiplexing and alignment to the human genome was performed using Illumina's CASAVA 1.8.2 software suite. The sample error rate was required to be less than 1% and the distribution of reads per sample in a lane was required to be within reasonable tolerance.
Quality control (QC) on MC-seq was conducted following standard procedure as previously described. Quality of sequence data was examined by using FastQC (ver. 0.11.8). Adapter sequences and fragments at 5’ and 3’ (phred score <30) with poor quality were removed by Trim_galore (ver. 0.6.3_dev). We used Bismark pipelines (ver. v0.22.1_dev) to align the reads to the bisulfite human genome (hg19) with default parameters(17). Quality-trimmed paired-end reads were transformed into a bisulfite converted forward strand version (C->T conversion) or into a bisulfite treated reverse strand (G->A conversion of the forward strand). Duplicated reads were removed from the Bismark mapping output and CpG, CHG and CHH (where H = A, T or C) were extracted.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *.bismark.cov: Tab-delimited text files: chromosome, start position, end position, percentage of methylation, methylated intensity, unmethylated intensity.
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| Submission date |
Jun 21, 2020 |
| Last update date |
Jun 23, 2020 |
| Contact name |
Ke Xu |
| E-mail(s) |
[email protected]
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| Phone |
2038240720
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| Organization name |
Yale University
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| Street address |
P.O. Box 208234.
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE152922 |
Comparison of Methylation Capture Sequencing and Infinium EPIC Methylation Array in Peripheral Blood Mononuclear Cells [MC-seq] |
| GSE152923 |
Comparison of Methylation Capture Sequencing and Infinium EPIC Methylation Array in Peripheral Blood Mononuclear Cells |
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| Relations |
| BioSample |
SAMN15338460 |
| SRA |
SRX8590966 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4629794_low-3C_251_326_S233_L003_R1_001_val_1_bismark_bt2_pe.deduplicated.bismark.cov.gz |
41.6 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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