| Autor: |
Castellano, Giancarlo, EVA GONZALEZ, Roset, Alba, Fernandez, Eva, Isanta, Ricard, Jimenez, Wladimiro, Jares, Pedro |
| Rok vydání: |
2017 |
| DOI: |
10.6084/m9.figshare.5729136.v1 |
| Popis: |
Next-generation sequencing is currently used in clinical settings for translational biomarker profiling and clinical research studies including inherited diseases and cancer. The Ion Ampliseq technology combined with the Ion Torrent PGM sequencer for somatic mutation identification is an easy to use, fast and cheap approach. We are routinely using the Oncomine Solid Tumour panel in our Service to study solid tumors using DNA fromformalin-fixed, paraffin embedded (FFPE) tissues. The Ion Ampliseq technology use a DNA barcodes system to provide flexibility and high-throughput capabilities in sequencing and to significantly increase scale while reducing costs by allowing to pool multiple library preparations in a single flow cell lane. Formalin fixation results in non-reproducible C>T/G>A sequencing artifacts that are observed after PCR amplification. We developed an optimized wet-bench workflow and a pipeline analysis to detect genomic variants based on the combination of two different barcodes for the same sample like in a duplicated assay. This design allows to increase the sensitivity and specificity of the sequencing test with the consequent reduction of false positive somatic variants due to artifacts. Moreover this new method allows to improve the detection of variant in regions with not optimal read coverage. Therefore we suggest an adjusted workflow for clinical diagnostic application that can help to detect genomic variants at low cost and with an improved performance with respect to the conventional procedure. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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