Whole Genome Bisulfite SequencingNext-generation Sequencing
Service OverviewDNA methylation is epigenetic code, which plays a vital role in regulating gene expression and influencing a variety of biological functions. Genome-wide profiling of methylation on cytosine could provide promising knowledge in decoding epigenetic markers. Bisulfite sequencing is a technology combines traditional methylation capture technique, high-throughput sequencing technology and bioinformatics, which enables efficient, accurate and affordable revealing of whole-genome cytosine methylation at single-base level.
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Nucleotide distribution test is designed to detect separation of AT and GC. According to DNA random fragmentation process and complementary base pairing principle, the frequency of A, T, G, C should be similar and steady along the reads without bisulfite treatment. In whole genome bisulfite sequencing, un-methylated cytosines (C) on genome are read as thymines (T) after bisulfite treatment. Therefore, frequency of T is increased while C is decreased on one strand and frequent of Adenine (A) is increased while Guanine (G) is decreased on the reverse strand.
Identification of 5mC is mainly processed by Bismark software. Un-methylated cytosines are transferred into uracils and are then transferred into thymine in PCR amplification. As a consequence, methylated cytosine remain unchanged and un-methylated cytosines are read as thymines. In corresponding reverse strand, guanines are read as adenines. Bismark is designed to convert these reads during mapping clean data against reference genome and determines the best match. By comparing the nucleotides on corresponding C sites on reference genome, 5mC can be identified.
5mC distribution on chromosome
Each chromosome is divided into 100 kb frames to evaluate methylation level within each frame and distribution of 5mC on chromosome-level. Whole genome 5mC distribution on chromosomes is presented on the map.
Highly methylated CGI region annotation
Methylation level higher than 0.7 is defined as highly-methylated CGI regions. C sites with sequencing depth higher than 5X are considered to be reliable. Excluding CGI regions with reliable C sites smaller than 0.1, the highly-methylated CGI regions are annotated. The promoter region is defined as 3,000 bp upper stream of a gene.
GO analysis on DMR related genes
GO classification analysis on DMR (differential methylated regions) related genes shows the DMR-based and all-gene-based enrichment of DMR related genes in each GO term and classification. It reveals potential functions and pathways affected by DMRs in terms of biological process, cellular component and molecular functions.
Signaling pathway analysis on DMR related genes
DNA methylation at certain sites are believed to affect the expression of corresponding genes, which further leads to different biological effects. Enrichment analysis of DMR related genes against GO and KEGG database enables the extraction of signaling pathways involved, which helps link potential DNA methylation markers to biological phenomena.
1How is the length of DMR defined? Say a the length of a DMR is 1 kb, are any fragments within this DMR still differential between groups?
Answer: DMR is identified by Bisulfighter software. The detailed process for DMR identification can be found in the paper below. (Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions, DOI：10.1093/nar/gkt1373) The length of DMR is not fixed. A fragment within a 1 kb DMR is not necessary a DMR as well. If several DMRs are found to be connected, the software will joint these DMRs into one DMR.
2How to calculate methylation level?
Answer: There are four methods of calculation. 1) Methylation level of single site: Methylation level of single site= number of reads support methyl-C/total reads Note: For C sites on reference genome: Methylation level of single site= number of reads support methyl-C (C in reads)/[number of reads support(C)+number of reads against (T)] 2)Methylation level of a region: [Fraction of methylated cytosines] Methylation level= number of methylated-C/ total number of C in the region 3)Methylation level of a region: [Mean methylation level] Methylation level= sum of methylation level on each single site/ total number of C in the region 4)Methylation level of a region: [Weighted methylation level] Methylation level= sum of reads supports all methyl-C within the region/ total number of reads within the region.