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Whole Genome Bisulfite Sequencing

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whole genome bisulfite sequencing
By Christoph Bock (Max Planck Institute for Informatics) (Own work) [CC BY-SA 3.0
(http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons
DNA methylation at the C5 position of cytosine plays a crucial role in gene expression and chromatin remodelling, and perturbations in methylation patterns are implicated in the development of cancer, neurodegenerative diseases, and neurological disorders. Therefore, the mapping of methylated bases (the methylome) is critical to understanding gene expression and other processes subject to epigenetic regulation.

Novogene provides whole genome sequencing of bisulfite-converted DNA, as an effective method to identify individually methylated cytosines on a genome-wide scale. Methylome analysis is an increasingly valuable research tool with a range of applications, including studies on gene regulation, stem cell differentiation, embryogenesis, aging, cancer and other diseases, and phenotypic diversity and evolution in plants and animals.

The Novogene Advantage

  • Extensive experience: over 400 samples successfully sequenced and a bioinformatics analysis team composed entirely of Ph.D. scientists.
  • Unsurpassed data quality: We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, exceeding Illumina’s official guarantee of ≥ 75%.
  • Comprehensive data analysis: We use widely-accepted mainstream software such as Bismark and a mature in-house pipeline for mapping, DMR analysis, functional analysis and data visualization.

Project Workflow

whole genome bisulfite sequencing project workflow

Sequencing Strategy

  • 250~300-bp insert bisulfite treated DNA library
  • HiSeq platform, paired-end 150 bp

Data Quality Guarantee

  • Over 80% of bases have a sequencing quality score ≥ Q30. See examples of our high quality data.

Sample Requirements

  • DNA amount: ≥ 5 μg
  • DNA concentration: ≥ 50 ng/μl
  • Purity: OD260/280 ≥ 1.8 ~ 2.0 without degradation or RNA contamination

Turnaround Time

  • 15 working days from verification of sample quality without data analysis
  • The data analysis turnaround is project-dependent.

Recommended Sequencing Depth

  • Sequencing depth > 30X

Analysis Pipeline

whole genome biosulfite sequencing analysis pipeline


Table. Representative samples showing data quality of Novogene’s whole genome bisulfite sequencing service.
Sample NameRaw ReadsRaw BasesClean ReadsClean BasesError Rate (%)Q20 (%)Q30 (%)GC (%)BS Conversion Rate (%)*
Sample 1750864918112.63 G686418034102.96 G0.0394.6286.5223.0199.29
Sample 2762188782114.14 G61849526092.61 G0.0494.3586.4121.0899.66
Sample 3789026912118.35 G721080112108.16 G0.0296.5490.6321.1299.66
Sample 4784535120117.68 G760489944114.07 G0.0392.8185.4920.7099.63

* BS, bisulfite

Project Example

The following study utilized Novogene's expertise in whole genome bisulfite sequencing.

Whole-Genome Analysis of 5-Hydroxymethylcytosine and 5-Methylcytosine at Base Resolution in the Human Brain
Genome Biology, 15: R49 (2014)

Different forms of DNA methylation may differentially modulate gene expression and chromatin structure, with consequences for organ development and function. There is particular interest in the role of 5-hydroxymethylcytosine (hmC), which is abundant in some types of mammalian cells. In this study, genome-wide sites of hmC and 5-methylcytosine (mC) in the human brain were mapped to single-base resolution using, respectively, bisulfite sequencing and Tet-assisted bisulfite sequencing. Strand biases were observed with higher levels of hmC and mC associated, respectively, with the sense and antisense strand of gene bodies. Levels of hmC increased during developmental stages, with highest levels present in adult brains, and hydroxymethylation-enriched sites included actively transcribed regions and 5’ splicing sites at exon-intron boundaries, indicating roles for hmC in the regulation of gene expression and splicing.

wgbs-data
Figure. Base-resolution hydroxymethylome and methylome in the human brain. (a) The percentages of hmCs or modCs in the adult or the fetal brain in the contexts of CG, CHH, and CHG. (b) Examples of the hmC, mC, and total modification (hmC + mC) profiles are shown for a genomic region of 12 Mb on chromosome 4 as a scatterplot (upper panel) and for a 12 kb region surrounding the TSS of the TET2 gene as a bar chart. The green box indicates the CpG island located in the TET2 promoter.

Publications Using Novogene’s Expertise

JournalTitle
Genome Biology, 15: R49 (2014)Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain.
Nature Communications, 3:850 (2012)An atlas of DNA methylomes in porcine adipose and muscle tissues.
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