Don’t amplify the noise...

Next generation sequencing (NGS) is now in routine use for a broad range of research and clinical applications. Facilitating the detection of a wide variety of mutations, focus has never been higher on the value of making the correct choice for the initial sequence enrichment step, which, if poorly designed, can be a source of bias and error in the downstream sequencing assay.

The two main enrichment strategies commonly used are amplicon (PCR)-based and hybridisation-based. Each has its own advantages and disadvantages. Amplicon has the advantage of offering a simpler workflow, it is faster, the ongoing cost of running an amplicon-based technology is relatively low and can accommodate a lower sample input. Hybridisation, on the other hand, is a more robust technique and has the advantage of providing better uniformity of coverage, fewer false positives, and superior variant detection due to fewer PCR cycles.

An important limitation of amplicon-based approaches is the difficulty with assessing the true complexity of the library – every molecule that you sequence will start and end with the same base (as determined by PCR primers used for Multiplex PCR) so it’s virtually impossible to remove duplicates without using molecular identifiers, which whilst excellent for deduplication and error correction, can reveal high levels of duplication and low complexity. This over representation of some regions, if those regions contain mutations,  either true or PCR induced errors, has the potential to impact downstream results.

Another disadvantage is fairly poor uniformity of coverage – certain regions of the genome will amplify better than others and therefore better depth of sequencing will be achieved in those regions, figure 1.