Methylation of DNA at cytosine nucleotides forms 5-methylcytosine (5mC), which impacts various cellular processes involving gene expression and chromatin remodeling. These processes can influence health and development through methylation of promoter regions, cell differentiation, remodeling of chromatin for selective X-chromosome inactivation, and suppression of transposable elements.
Quick Biology is providing various types of standardized and customized methylation sequencing services. Whole genome bisulfite sequencing (WGBS) is an effective and reliable strategy to identify individually methylated cytosines on a genome-wide scale. As an alternative, we can focus on the detection of DNA methylation to a specific subset of the genome using Reduced Representation Bisulfite Sequencing (RRBS) that combines restriction enzyme digestion with bisulfite sequencing to enrich for a CpG-dense fraction of the genome and profile DNA methylation at single-nucleotide resolution. By enriching for CpG-dense regions and sequencing only the fragments pertaining to those regions, the RRBS platform allows for the capture of a significant amount of methylation data while reducing the amount of sequencing, leading to a substantially decreased cost. This combination makes RRBS the perfect platform for pilot studies. While bisulfite sequencing has been the gold standard for methylome analysis, this conversion treatment damages DNA, resulting in fragmentation, loss, and bias. In contrast, Quick Biology now provides a high-performance and a highly effective enzyme-based alternative method called "Enzymatic Methyl-seq" (EM-seq) instead of bisulfite conversion for methylome analysis using Illumina® sequencing for superior detection of 5-mC and 5-hmC from fewer sequencing reads.
For analysis of genome-wide 5-hydroxymethylcytosine (5-hmC) positions at single-base resolution, Quick Biology can provide a semi-quantitative method as the level of 5hmC, called Reduced Representation Hydroxymethylation Profiling (RRHP 5-hmC) sequencing. For interrogation of both 5hmC and 5mC, and accurately quantifying the true level of cytosine methylation, we can provide the Ovation Oxidative bisulfite sequencing (oxBS-Seq) using Ultralow Methyl-Seq with TrueMethyl® oxBS or RRBS Methyl-Seq System.
Benefits of Quick Biology Whole Genome and Reduced Representation Bisulfite Sequencing (WGBS & RRBS):
- - Enabled genome-wide or a subset analysis of 5mC nucleotides at single-nucleotide resolution
- - Accommodates ultra-low DNA input and compatible with FFPE samples
- - Ideal for methylation analysis of precious, limited, and target-enriched samples
- - Compare differentially methylated loci from different samples
Quick Biology WGBS Workflow:
Quick Biology RRBS Workflow:
Benefits of Quick Biology EM-seq for methylation analysis:
- - Superior sensitivity of detection of 5-mC and 5-hmC, Greater mapping efficiency, and More uniform GC coverage
- - Detection of more CpGs with fewer sequence reads, lower duplication rates, and Uniform dinucleotide distribution
Quick Biology EM-seq Workflow:
Libraries are prepared using as little as 10 ng input DNA with the optimized EM-seq workflow in which TET2 oxidizes 5-mC and 5-hmC, providing protection from deamination by APOBEC in the next step. In contrast, unmodified cytosines are deaminated to uracils. Libraries are then amplified and sequenced using Illumina instrumentation.
Quick Biology oxBS-seq Workflow:
Researchers interested in quantitating 5hmC can use Quick Biology's TrueMethyl oxBS-seq to process samples in parallel preparations of oxBS and bisulfite-only to determine the 5hmC content through subtractive analysis methods (Figure below). The streamlined workflow consists of five main steps: fragmentation of genomic DNA, end repair to generate blunt ends, adaptor ligation, optional oxidation for 5-hmc (left figure), bisulfite conversion, and PCR amplification to produce the final library. With the TrueMethyl oxBS Module, parallel workflows with and without oxidation can be performed for analysis of 5hmC. The entire workflow, including fragmentation, can be completed in less than two days and generates DNA libraries ready for single read or paired-end sequencing.
Sample Requirements For WGBS, RRBS, EM-seq, and oxBS-seq:
- - Human, animals, plants, and microorganisms
- - Tissue, Whole Blood, Buccal Swab, Saliva, and Cell lines
- - FFPE: Contact for details and quality requirements
- - Genomic DNA (DNA can be 10 pg - 500 ng; OD260/280=1.8~2.0; free of enzymatic inhibitors)
- - Amount: Minimum 500ng DNA Recommended
- - Illumina Platform: Hiseq or NovaSeq, PE 150, 330-375M for WGBS, 25-30M for RRBS, and 100-200M read pairs per sample for EM-seq.
- - Estimated Coverage for WGBS, RRBS, EM-seq, and OxBS-seq: 5-25X
Quick Biology WGBS, RRBS, EM-seq and oxBS-seq Data Analysis:
- - Raw reads preprocessing, Remove adapters, and QC
- - Read Mapping to reference genome, Remove PCR bias and QC
- - Whole Genome Methylation Calling
- - Differential Methylation Analysis and Annotations
- - Results as tables, BED files, and summary statistics