The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences.

The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts.

Evaluation of a Four-Gene Panel for Hereditary Cancer Risk Assessment.

BRCA1/2 are tumor suppressor genes involved in DNA double-strand break repair. They are the most penetrant genes for hereditary breast and ovarian cancers, but pathogenic variants in these two genes can be identified only in a fraction of hereditary cases. Following the diffusion of BRCA molecular testing and the availability of specific therapeutic strategies for the management of pathogenic variant carriers, the demand for the analysis of additional predisposing genetic factors has increased. Indeed, there is accumulating evidence regarding the role of other genes, including CHEK2 and PALB2. Both of them are involved in the same molecular pathway as BRCA genes, with CHEK2 being responsible for cell cycle stopping to allow the repair of DNA double-strand breaks and PALB2 being able to interact with BRCA1 and activate BRCA2. Thus, their role as additional hereditary cancer predisposing factors is intriguing. Accordingly, guidelines for hereditary cancer risk assessment have been updated to include the criteria for additional genes testing. In this context, we validated a commercially available kit allowing for the simultaneous analysis of BRCA1, BRCA2, CHEK2 and PALB2. Forty-eight patients, already tested for BRCA mutational status, were re-analyzed in the present study. Results comparison showed that the tested method was able to correctly identify all the variants previously detected in the same patients. In particular, all single-nucleotide variants and small indels were correctly identified. Moreover, two copy number variants, included to assess the software's performance in detecting this kind of gene alteration, were also detected. Even if copy number variant estimation still requires confirmation by a molecular technique to avoid false positive results, it is able to reduce the number of patients requiring multiplex ligation probe amplification analysis, positively impacting the test's turnaround time. Finally, since the time and costs of the analysis are similar to those required just for BRCA genes, this strategy may be affordable for providing a more comprehensive test for hereditary cancer risk assessment.