Experts in bisulphite sequencing
Unveil the DNA methylation patterns of your study species
Bisulphite sequencing is a technique used to determine the pattern of DNA methylation, which is related to gene expression. Bisulphite treatment converts cytosines into uracils, but leaves methylated cytosines unaffected. As a result, all the cytosines detected in the high-throughput sequencing data correspond to methylated cytosines.
At AllGenetics we carry out whole-genome bisulphite sequencing (WGBS) and reduced-representation bisulphite sequencing (RRBS) protocols. While WGBS detects methylated cytosines all throughout the genome, RRBS uses a restriction enzyme to enrich the libraries in CpG contexts, where most of the DNA methylation occurs.
How we work
Step 1: We isolate DNA from your samples. We have adapted different DNA isolation protocols, depending on the starting biological material.
Step 2: We prepare genomic libraries compatible with Illumina’s HiSeq platforms. We carry out either WGBS or RRBS protocols in which DNA is treated with bisulphite. In the case of RRBS, a restriction enzyme is used to enrich the libraries in CpG contexts.
Step 3: We sequence the libraries using the Illumina HiSeq technology. Millions of reads per library are obtained in this step.
Step 4: We analyse the high-throughput sequencing data obtained in the previous step.
For your convenience, we can carry out the entire project or only those steps you require. As a norm, steps 2 and 3 should be ordered jointly. Please contact us for further information and pricing.
What you receive
A report with a summary of the methods followed.
The raw high-throughput sequencing files.
The results of the bioinformatic analyses including demultiplexing, pre-processing and quality control of the raw data, bisulphite-sensitive read alignment against the reference genome, deduplication of clonal reads (WGBS) or quality filtering by restriction sites (RRBS), methylation calling, and detection of differentially methylated positions (DMPs) and regions (DMRs) among conditions.
If you require additional analyses, please let us know and we will do our best to meet your needs.
At no additional cost, our project specialists will be available to you after project delivery and during the editorial process of your scientific articles. We offer up to five 60-min sessions per project, depending on the project size.
What we need
We can start working by using either your tissue samples, or DNA which has already been extracted.
If you are sending tissue samples, please make sure that they have been properly preserved for DNA isolation (ethanol, silica gel, frozen, or another suitable preservation method). If required, we can provide sampling kits and sample collection guidelines to ensure that your samples arrive at our lab in optimal conditions.
If your DNA has already been extracted, we ideally require 1 μg of DNA in TE buffer or water at a minimum concentration of 25 ng/μL, and 260/280 and 260/230 absorbance ratios higher than 1.8.
Please note that, depending on the specifics of your project, these requirements may vary. Please contact us for the specific requirements of your project.