Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Sin- gle-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 Sequenc- ing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key fac- tors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplifi- cation (WGA) method, for low-pass genome sequencing with the Ion TorrentTM platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on sin- gle 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 sin- gle 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 Ampli1TM WGA-based low- pass workflow for detection of CNAs in single tumor cells which would be of particular inter- est for genome-driven targeted therapy selection and for monitoring of disease progression.

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products.

Del Monaco V;Terracciano M;Medoro G;Rihawi K;Ardizzoni A;Manaresi N
2018

Abstract

Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Sin- gle-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 Sequenc- ing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key fac- tors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplifi- cation (WGA) method, for low-pass genome sequencing with the Ion TorrentTM platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on sin- gle 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 sin- gle 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 Ampli1TM WGA-based low- pass workflow for detection of CNAs in single tumor cells which would be of particular inter- est for genome-driven targeted therapy selection and for monitoring of disease progression.
Ferrarini A, Forcato C, Buson G, Tononi P, Del Monaco V, Terracciano M, Bolognesi C, Fontana F, Medoro G, Neves R, Möhlendick B, Rihawi K, Ardizzoni A, Sumanasuriya S, Flohr P, Lambros M, de Bono J, Stoecklein NH, Manaresi N
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/678760
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