Pyrosequencing Service Details
Project and Assay Design Workflow
We will first have a meeting to discuss your research goals and the suitability of the Pyrosequencing technology in achieving those goals. If Pyrosequencing is applicable, we’ll need you to provide bioinformatic data (e.g., annotated sequences, genomic coordinates) for your regions of interest, including marking the exact sites of variation that would like to assay. From that sequence, we’ll use Qiagen’s Assay Design software to design PCR and pyrosequencing primers, and will optimize the assay using our internal human and mouse controls. If your lab has samples of known genotype or methylation status, we may ask for aliquots of those samples during the validation stage.
Sample Processing Workflow
Once gDNA samples are received, LMT staff will determine the concentration and total DNA via NanoDrop. An aliquot of the sample will be normalized to 10 ng/uL, and 30 ng will be used as template for the Pyrosequencing PCR. The Pyrosequencing reaction will consume ~ half of the PCR product generated, which will allow for the sample to be repeated once in the event of poor quality data and per the customer’s request. Data will be analyzed using Qiagen’s genotyping analysis software, and an Excel document with genotype data and a Word document with each samples’ Pyrogram will both be sent electronically to the customer.
Sample Processing Workflow (Bisulfite Conversion and DNA Methylation)
Once gDNA samples are received, LMT staff will determine the concentration and total DNA via NanoDrop. 100 ng of gDNA will be used a template (as well as vendor supplied controls) for the bisulfite conversion reaction using Qiagen’s EpiTect Bisulfite Kit, followed by purification and handling of the converted DNA (“bcDNA”). Although ~90% of the gDNA is degraded during the harsh sodium bisulfite treatment, the amount of converted DNA template collected and purified is sufficient for ~ 100 bisulfite PCR amplifications. Following the Pyrosequencing sample workflow, data will be analyzed using Qiagen’s CpG analysis software, and an Excel document with methylation levels at each CpG site and a Word document with each samples’ Pyrogram will both be sent to electronically the customer.