In order to simplify the sequencing process and to reduce the costs associated with individual labelling of DNA samples, we have developed a mutation detection approach based on targeted NGS in combination with high resolution melting (HRM) analysis.
We used a somatic mutation detection approach, since this is the most suited for the detection of variants represented in very low frequency (1 in 32 alleles, in the lowest case).
The technique is based on the mutation detection approach used in TILLING (Targeting Induced Local Lesions in Genomes) [ 7, 8].
Traditional approaches using genetic linkage or candidate gene analysis have often been limited, costly, and slow to yield new insights, but the advent of next-generation sequencing (NGS) technologies has altered the landscape of current gene discovery and mutation detection approaches.
Unfortunately, there is an almost universal lack of validation of clinically relevant mutation detection approaches in clinical settings against a sensitive gold standard approach.
Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking.
In this study, we have validated two sequencing-based mutation detection approaches (dideoxy- and pyrosequencing) against parallel sequencing in a clinical setting, taking into account the tumor cell content as well as the quality and the type of tissue (e.g. FF, FFPE) of each specimen.
Several alternative ultra-sensitive mutation detection approaches have been proposed recently for next generation sequencing [ 15, 31, 32], but they require significant sample preparation and may not be practical beyond targeting limited regions of a genome.
However, in these cases, it is current practice to establish phase using single sperm cells in affected males, or simply to use linked markers as a backup to direct mutation detection, and the same approaches could be used with karyomapping.
Hence, whilst the use of microarrays and NGS in genomic analysis will likely continue to be complementary [ 10], we here describe work specifically aimed at optimizing genomic deletion mutation detection via microarray-based approaches.
The most well-known NGS technologies have been developed by Illumina (and Solexa, Inc, purchased by Illumina in 2007; San Diego, CA, USA), Helicos BioSciences (Cambridge, MA, USA), ABI/SOLiD, and 454 Life Sciences (a subsidiary of Roche; Branford, CT, USA) and use a single-molecule template for mutation detection with cloning-free approaches.
