Maria De Bonis

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

BRCA1/2 screening in Hereditary Breast and Ovarian Syndrome (HBOC) is an essential step for effective patients’ management. Next-Generation Sequencing (NGS) can rapidly provide high throughput and reliable information about the qualitative and quantitative status of tumor-associated genes. Straightforwardly, bioinformatics methods play a key role in molecular diagnostics pipelines. BRCA1/2 genes were evaluated with our NGS workflow, coupled with Multiplex Amplicon Quantification (MAQ) and Multiplex Ligation-dependent Probe Amplification (MLPA) assays. Variant calling was performed on Amplicon Suite, while Copy Number Variant (CNV) prediction by in house and commercial CNV tools, before confirmatory MAQ/MLPA testing. The germline profile of BRCA genes revealed a unique HBOC pattern. Although variant calling analysis pinpointed heterozygote and homozygote polymorphisms on BRCA1 and BRCA2, respectively, the CNV predicted by our script suggested two conflicting interpretations: BRCA1 duplication and/or BRCA2 deletion. Our commercial software reported a BRCA1 duplication, in contrast with variant calling results. Finally, the MAQ/MLPA assays assessed a whole BRCA2 copy loss. In silico CNV analysis is a time and cost-saving procedure to powerfully identify possible Large Rearrangements using robust and efficient NGS pipelines. Our layout shows as bioinformatics algorithms alone cannot completely and correctly identify whole BRCA1/2 deletions/duplications. In particular, the complete deletion of an entire gene, like in our case, cannot be solved without alternative strategies as MLPA/MAQ. These findings support the crucial role of bioinformatics in deciphering pitfalls within NGS data analysis.