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.

Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics.

Fazio V. M.
2019-01-01

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12610/895
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