• Alberto Ferrarini, Claudio Forcato, Genny Buson, Paola Tononi, Valentina Del Monaco, Mario Terracciano, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Rui Neves, Birte Möhlendick, Karim Rihawi, Andrea Ardizzoni, Semini Sumanasuriya, Penny Flohr, Maryou Lambros, Johann de Bono, Nikolas H Stoecklein, and Nicolò Manaresi.
• Menarini Silicon Biosystems spa, Bologna, Italy.
• Plos One. 2018 Jan 1; 13 (3): e0193689.

AbstractChromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.

29 keywords...

hide…