When conducting a Next-Gen sequencing experiment, the quality of the starting material is of utmost importance. Often, DNA or RNA extraction protocols that are sufficient for applications such as PCR, Cloning, Southern or Northern blots are unable to generate material that is of high enough quality for sequencing. For DNA applications it is important that the DNA start out at a certain size in order to fragment effectively and generate libraries with high complexity. It must also be free of PCR inhibitors, salts and organic solvents which can affect downstream enzymatic processes. Many of the same criteria apply to RNA which is more susceptible to degradation.
To generate DNA or RNA of high quality it is important to use lysis methods that are appropriate for the cell type. The lysis should ideally be strong enough to break cells open but not so harsh that it chews up the nucleic acids. It is also important to work in a nuclease free environment to prevent degradation of material. The best way to avoid problems with nucleases is to work quickly and avoid prolonged incubations after breaking open the cells. For genomic DNA, it is also important to avoid vortexing the sample and to use wide bore tips to prevent mechanical shearing of the DNA. Many of these sample quality details are outlined in our DNA and RNA sample reference guides.
At Cofactor, we make sure your experiment starts out with the best possible starting material. Samples undergo various layers of QC before the final product is passed on to sequencing. Based on the specifics of the project, samples are analyzed by nanodrop, fluorescence quantification measurements, and fragment analysis on the bioanalyzer. Our experience with processing thousands of these samples has given us a pretty good eye for the QC parameters that work best for sequencing. Based on these initial results we provide you with feedback to help determine if sample quality is sufficient to proceed to library construction or whether we need to evaluate the extraction process. We are more than happy to offer our expertise to help find an extraction protocol that will work best for your needs and are often able to handle the extractions ourselves to simplify your workflow. Contact us if you’d like more information on our sample extraction offerings.
