Revista de genómica clínica y médica

Targeting Cancer Related Genes by Multiplex PCR Followed by High Throughput Parallel Sequencing

Abstract

Vollbrecht C, Mairinger F, Schweighofer CD, Heukamp L, Merkelbach-Bruse S, Büttner R and Margarete O

The detection of a wide range of genomic alterations plays an important role in the diagnostics improving individual therapeutic approaches of cancer patients. Technologies that help to identify therapeutic relevant targets in tumor samples are a major factor on the way to a personalized medicine. The number of predictive and prognostic markers that influence the therapeutic outcome is continuously increasing. Therefore, parallel sequencing also named next generation sequencing (NGS), allowing the simultaneous analysis of numerous cancer related hotspots in many patients starting from a limited amount of DNA, is urgently needed to be established in cancer diagnostics. Different methods of target library preparation are commercially available and offer the opportunity to sequence tumor relevant hotspot mutations in a panel of genes.

In the present study, a multiplex PCR approach, targeting a lung cancer and a leukemia gene panel, each consisting of 20 disease and therapy relevant genes, was investigated. Twelve formalin fixed and paraffin embedded lung tumors and twelve native Chronic Lymphocytic Leukemia (CLL) samples, respectively, were analyzed. Samples were sequenced on the MiSeq sequencer platform.

The results showed a very high quality of each run. In spite of the low DNA input, each multiplex approach allowed a simultaneous analysis of 20 genes, in total covered by around 1,000 amplicons, in up to twelve cancer samples.

Thus, the application of NGS on amplicon targets revealed an excellent performance in detecting a wide range of genetic alterations, combined with a high sensitivity.