Sequence - Am I dealing with the right genotype?

The sequence of a nucleic acid fragment is the most valuable and insightful piece of information a sample can deliver. Sequencing a sample is the ultimate QC step scientists should perform to verify and validate that the pieces of DNA or RNA they are working with are the correct ones and that the genetic information has not been altered in any way along the workflow.

Sequencing technology such as Pyrosequencing not only provides sequence information, but can also detect biochemical alterations such as DNA methylation and quantify occurrence of sequence variants within a DNA population. For QC purposes, targeted sequencing is most commonly performed, targeting one or more specific regions important to the success of downstream applications and for interpretation of the information from the sequence.

Sequencing provides scientists with reads from which the sequence of A, C, T, G (and U for RNA) can be deduced. Reads can be assembled and aligned against a reference sequence in order to highlight differences and similarities. The outcome of this alignment lets you know if the sample you are analyzing is the right one. This gatekeeper QC step prevents you from processing incorrect samples or samples of poor quality, thus reducing the risk of misinterpreting the final data due to the presence wrong or altered samples.

In addition, the high sensitivity and quantification features of some sequencing technologies can help highlight a variant sequence among a broader population of nucleic acid samples or alert you to the presence of contaminating sequences in a sample assumed pure. In the first case, sequencing can be used to find and quantify rare mutations including SNPs in complex samples, while in latter case, sequencing can offer qualitative insight regarding the sample purity.

Before asking what your results mean, check that you are working with the right variant! Sequence verification is of highest importance when trying to link an observed phenotype to a genotype or to highlight mutations or variants that could explain an observed phenotype.

There are currently three main technologies commonly used for DNA sequencing: Sanger sequencing, Pyrosequencing and next-generation sequencing (NGS). NGS is an umbrella designation for several different technologies used for massively parallel sequencing, enabling high-throughput sequencing of multiple samples at the same time, with high coverage.

Pyrosequencing is the technology of choice for quality control, since it is both a qualitative and quantitative method that enables rapid and accurate quantification of sequence variation. Streamlined protocols, analysis flexibility and elegant output make Pyrosequencing a highly adaptable tool for research in a broad range of disciplines.