Sorting was realized on the basis of morphological and phenotypic features of MDA/MCF-7 cells: they presented an oval shape and round features and high nuclear–cytoplasmic ratio.
MDA-MB-231 (MDA) and MCF-7 cell lines were employed as surrogate Breast Cancer (BC) CTCs and used in spiking-in experiments by addition to a healthy donor blood sample before DEPArray TM isolation. Cell Detection and DNA Integrity Assessment The original protocol was adapted for NGS analysis of few pooled cells isolated on a DEPArray TM system, with the overall aim of completely skipping WGA instead, directly proceeding with library construction while obtaining reliable sequencing results.Ģ.1.1. This approach is capable of intercepting even single DNA molecules in difficult biological samples, such as ctDNA. This work proposes a modified workflow for a targeted Next-Generation Sequencing (NGS) panel, which already was tested in our laboratory for the molecular characterization of Circulating Tumor DNA (ctDNA) and which is based on molecular tagging technology. Meanwhile, different workflows have been developed to provide alternative options to the traditional techniques which do not require this preamplification step, instead of utilizing downstream whole-genome or targeted sequencing. These biases are partly caused by an unavoidable delay in the mutational profile analysis of CTCs in routine laboratory practice.
#Final draft tagger torrent series
Unfortunately, different studies have demonstrated a series of drawbacks arising from WGA procedures, such as allelic dropout, chimeric molecules, low uniformity, and polymerase errors, which lead to bias in the interpretation of molecular data. To date, major efforts towards the molecular characterization of CTCs have been focused on the optimization of genetic analysis protocols, mainly including Whole Genome Amplification (WGA) as a first step, which allows for the isolation of an acceptable amount of DNA from few starting molecules. Īn increasing number of data have confirmed the role of CTCs count and their molecular characterization in patient prognosis, relapse prediction, and therapy outcome, indicating that CTCs represent an attractive blood-based biomarker with high potential for understanding the clinical behavior of the associated disease. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.Ĭirculating Tumor Cells (CTCs) in the bloodstream, which originate from primary and/or metastatic tumors, are both phenotypically and molecularly highly heterogeneous. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA).
0 kommentar(er)
