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|Public on Mar 18, 2020
|subject id: SUB74
cancer status: non-cancer
library: ACCEL-NGS Methyl-Seq DNA Library
|DNA extracted using phenol-chloroform protocol after RNAse treatment. DNA was bisulfite converted with the EZ DNA methylation kit (Zymo).
For Nextera transposase-based library approach, genomic DNA was first tagmented using Nextera XT transposome and end repair was performed using 5mC. After bisulfite conversion, Illumina adapters and custom bisulfite converted adapters are attached by limited cycle PCR. Illumina TruSeq DNA Methylation Kit and ACCEL-NGS Methyl-Seq DNA Library kit were used according to the manufacturer’s instructions.
|Illumina NovaSeq 6000
|Basecalls was performed using CASAVA
After trimming for low-quality bases (Phred score<30) and reads with a length <40 bp with TrimGalore, the reads were aligned to the human genome (GRCh37) using Bismark with paired end mode and default setting . For ACCEL-NGS Methyl-Seq DNA Library, as recommended by the manufacturer, 15 bp were trimmed on 5' and 3' of both reads.
Duplicate reads were removed using Picard tools and reads with more than 10% unconverted CHG or CHH cytosines were filtered out
SNP calling was performed with BisSNP using default settings, except for the maximum coverage filter set at 200. Heterozygous SNPs with less than 5 reads per allele were filtered out. In addition, SNP with multiple mapping positions were filtered out, as well as SNPs with more than one minor allele with allele frequency>0.05. or deviating significantly from Hardy-Weinberg equilibrium based on exact tests corrected for multiple tests. C/T and G/A SNPs were assessed after filtering out reads mapping to the C/T strand.
ASM calling was performed after separating the SNP-containing reads by allele using R. After Bismark methylation extractor is applied, CpG methylation calls by allele are retrieved using allele tagged read IDs. Paired reads with ambiguous SNP calling (i.e., called as REF allele on one paired end and ALT allele on the other) were discarded.
genome build: GRCh37
processed data files format and content: Processed files per sample contain difference in methylation percentage at the CpG level between the alternate and reference alleles for each SNPs associated with high confidence ASM (observed in >2 biological samples) if the given sample was informative. The columns include chromosome, start and end of the CpG (reported on positive strand), difference in methylayion percentage and SNP ID. The unique identifier in these files is the CpG coordinate AND SNP ID.
|Sep 23, 2019
|Last update date
|Mar 20, 2020
|40 prospect avenue
|Whole Genome Bisulfite sequencing: Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs
|Whole genome bisulfite sequencing and Genome-wide targeted methyl-seq: Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs
|SRA Run Selector