Reduced Representation Bisulfite Sequencing (RRBS) Service – Accredited DNA Methylation Profiling for Global Epigenomics Research
Our internationally accredited laboratory delivers a specialist reduced representation bisulfite sequencing (RRBS) service that provides biomedical researchers, pharmaceutical developers, academic institutes, clinical‑diagnostics companies and agrigenomics enterprises worldwide with the precise, reproducible and base‑resolution DNA methylation data they need to unravel the epigenetic regulation of health, disease and development. Every project is executed under the rigorous framework of ISO/IEC 17025, and each report bearing the ILAC mark is unconditionally accepted by regulatory authorities, granting bodies and supply‑chain partners in all major economies. The RRBS sulfite sequencing platform enriches the CpG‑rich regions of the genome by the MspI restriction enzyme digestion, performs the bisulfite conversion of the unmethylated cytosines to uracils, and sequences the resulting libraries on the Illumina NovaSeq or the comparable high‑throughput platforms. By focusing the sequencing capacity on the biologically most informative fraction of the methylome, this rrbs sulfite sequencing service delivers the quantitative methylation status of millions of individual CpG sites across the promoters, the CpG islands, the shores and the gene bodies at a fraction of the cost of the whole‑genome bisulfite sequencing, making it the method of choice for the large‑scale, population‑based epigenome‑wide association studies, the biomarker‑discovery cohorts and the drug‑response‑screening programmes.
Product Samples We Regularly Accept for RRBS Sulfite Sequencing
The nucleic‑acid extraction, quality‑control, bisulfite‑conversion and library‑preparation suites in our facility are equipped to process a broad diversity of sample types and input quantities. The following categories represent the most frequently analysed specimens:
- Mammalian tissues and biopsies – fresh‑frozen and the formalin‑fixed, paraffin‑embedded (FFPE) sections of the tumour, the normal adjacent tissue, the brain, the liver, the placenta and the other organs, with the DNA extracted by the optimised protocols that minimise the further fragmentation of the already degraded FFPE DNA
- Whole blood, buffy coat and the peripheral‑blood mononuclear cells – the samples collected into the EDTA, the citrate or the PAXgene tubes, which provide the high‑molecular‑weight genomic DNA that is ideal for the MspI digestion and the bisulfite conversion
- Cell lines and the in‑vitro cultured primary cells – the cultured cancer cell lines, the induced‑pluripotent‑stem‑cell lines, the embryonic‑stem‑cell lines and the primary fibroblasts, keratinocytes and the immune‑cell subsets, for which the methylation profiles before and after the drug treatment, the differentiation or the gene‑editing are compared
- Laser‑capture microdissected and the flow‑sorted cell populations – the low‑input samples from the microdissected tumour sub‑regions, the sorted neuronal nuclei, the stem‑cell niches and the rare circulating tumour cells, processed with the ultra‑low‑input library‑preparation workflows that require as little as 10 ng of the genomic DNA
- Plant and the animal tissues for the agrigenomics and the ecology studies – the leaf, the root, the seed, the insect, the fish and the livestock tissues, where the CpG and the non‑CpG methylation patterns are analysed to link the epigenetic variation to the agronomic traits and the environmental adaptation
- Plasma‑derived cell‑free DNA and the circulating tumour DNA – the low‑concentration, highly fragmented DNA from the liquid‑biopsy samples, for which the RRBS library‑preparation protocol is adapted to capture the methylation information from the short DNA fragments that are characteristic of the apoptotic and the necrotic cell death
Library Preparation, Bisulfite Conversion and Sequencing – The Core Workflow of the RRBS Sulfite Sequencing Service
- Enzymatic digestion with the MspI restriction endonuclease according to the internal validated protocol and the principles of the original Meissner and the Boyle RRBS procedures: the high‑molecular‑weight genomic DNA is quantified by the fluorometry and the integrity is assessed by the capillary electrophoresis. An aliquot of 100 ng to 500 ng of the DNA is digested with the MspI enzyme, which recognises the 5′‑CCGG‑3′ sequence and cuts the DNA regardless of the methylation status, generating the fragments that are enriched in the CpG‑rich regions. The digestion efficiency is verified by the electrophoretic analysis of a control DNA, and the digested DNA is purified, end‑repaired, A‑tailed and ligated to the methylated adaptors that carry the unique dual‑index barcodes for the sample multiplexing. This rrbs sulfite sequencing step captures the CpG‑dense fraction of the genome that contains the vast majority of the functionally relevant methylation sites, while the CpG‑poor, intergenic and the repetitive regions that are less informative are largely excluded from the library.
- Bisulfite conversion and the final library amplification according to the internal validated protocol and the best‑practice guidelines of the International Human Epigenome Consortium: the adaptor‑ligated DNA fragments are treated with the sodium bisulfite under the conditions that convert the unmethylated cytosines to uracils while leaving the methylated cytosines unchanged. The conversion efficiency is monitored by the inclusion of the unmethylated lambda‑phage DNA or the other spike‑in control, and a conversion rate of at least 99.5 % is required for the library to proceed to the sequencing. The bisulfite‑converted, single‑stranded DNA is then amplified by the polymerase chain reaction with the minimum number of the cycles that yield the sufficient library quantity, minimising the PCR‑duplication rate and the GC‑bias. The final library is quantified, and the fragment‑size distribution is checked on a Bioanalyzer or a TapeStation system, and the libraries are pooled in the equimolar ratios for the sequencing.
- High‑throughput sequencing on the Illumina NovaSeq or the HiSeq platforms: the pooled library is sequenced in the paired‑end mode – typically 2 × 150 base‑pairs – to a depth that provides the adequate coverage of the CpG sites across the reduced‑representation genome. The sequencing‑depth recommendations depend on the genome size and the desired statistical power: for the mouse and the human genomes, 20 million to 40 million read‑pairs per sample are typically sufficient to cover 1 million to 2 million CpG sites with a depth of at least 10×, enabling the reliable methylation quantification at the single‑CpG resolution. The raw sequencing data are demultiplexed, and the quality of the run is assessed by the Q30 score, the per‑base sequence content and the duplication rate, and the data are delivered as the FASTQ files.
Bioinformatics Analysis and Methylation Quantification – Data Delivery from the RRBS Sulfite Sequencing Experiment
- Read alignment and the methylation‑calling pipeline according to the internal validated workflow using the Bismark, the BSMAP or the BS‑Seeker2 software: the raw reads are trimmed of the adaptor sequences and the low‑quality bases, and the trimmed reads are aligned to the reference genome – typically the GRCh38 for the human, the GRCm39 for the mouse, or the customer‑specified assembly – using the bisulfite‑aware alignment algorithm that simultaneously accounts for the C‑to‑T conversion on the forward reads and the G‑to‑A conversion on the reverse reads. The uniquely aligned reads are retained, and the methylation state of each cytosine in the CpG, the CHG and the CHH sequence contexts is extracted, with the bisulfite‑conversion efficiency estimated from the non‑CpG cytosines of the lambda‑genome spike‑in or the mitochondrial DNA. The methylation ratio at each covered cytosine is reported as a percentage, and the summary statistics – the total number of the CpG sites covered, the mean coverage depth per CpG, and the global average CpG methylation level – are provided for each sample.
- Differential‑methylation analysis between the experimental groups according to the internal validated workflow using the methylKit, the DSS or the RADMeth software: the CpG sites that are covered in all the samples of the comparison are tested for the differential methylation between the groups – for example, the tumour versus the normal, the treated versus the untreated, or the responder versus the non‑responder – using the logistic‑regression or the beta‑binomial model that accounts for the biological variability and the coverage depth. The differentially methylated CpG sites and the differentially methylated regions are reported with the methylation‑difference, the adjusted p‑value and the genomic annotation, and the results are visualised as the volcano plots, the heatmaps and the chromosomal‑distribution plots, providing the comprehensive, publication‑ready output that the biologist can directly interpret.
- Annotation of the differentially methylated regions to the genomic features and the gene‑set enrichment analysis: the differentially methylated regions are annotated to the nearest gene, the promoter, the CpG island, the enhancer and the other genomic regulatory elements using the HOMER, the GREAT or the ChIPseeker tools, and the genes that are associated with the differentially methylated regions are tested for the enrichment of the Gene‑Ontology terms, the KEGG pathways and the Reactome pathways, providing the functional interpretation of the methylation changes. This rrbs sulfite sequencing analysis links the epigenetic alterations to the biological processes and the disease pathways, which is the key deliverable for the grant‑funding, the publication and the regulatory‑submission purposes.
- Integration of the RRBS methylation data with the RNA‑seq or the other omics datasets: for the studies that have generated the transcriptomic, the genomic or the proteomic data from the same samples, the correlation between the promoter methylation and the gene expression, or the impact of the genetic variants on the methylation quantitative trait loci, is analysed, providing the multi‑omics, systems‑biology perspective that is increasingly required by the high‑impact journals and the precision‑medicine initiatives.
- Quality‑control reporting and the data‑delivery formats: the complete quality‑control report – including the per‑sample bisulfite‑conversion efficiency, the M‑bias plot, the CpG‑coverage distribution and the sample‑clustering by the principal‑component analysis of the methylation data – is delivered alongside the methylation‑ratio tables in the text or the bedGraph format, the raw FASTQ files and the aligned BAM files, allowing the customer to reproduce the entire analysis or to perform the custom downstream processing.
Report Acceptance and Global Regulatory Compliance
All rrbs sulfite sequencing projects are executed under the fully accredited scope of our ISO/IEC 17025 quality management system. Each project report that carries the ILAC mark is therefore automatically recognised by regulatory authorities, research‑funding bodies, notified bodies and supply‑chain partners in all major economies. For pharmaceutical researchers, academic epigenomics laboratories, clinical‑diagnostics developers and agrigenomics enterprises anywhere in the world, the report constitutes legally robust, internationally accepted evidence that the DNA‑methylation profiling, the differential‑methylation analysis and the functional annotation have been performed in accordance with the internal validated protocols and the customer‑specified methods. The documentation can be directly used to support the investigational‑new‑drug application, the biomarker‑patent filing, the publication in the peer‑reviewed journal, the issue of inspection certificates according to EN 10204 or equivalent national standards, and the resolution of commercial and technical disputes concerning the quality, the reproducibility and the biological interpretation of the reduced‑representation bisulfite‑sequencing data.
