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

PloS One
Alberto FerrariniNicolò Manaresi

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. 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 p...Continue Reading

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Citations

Dec 15, 2018·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Mariano Di TrapaniGianni Medoro
Jul 8, 2018·Clinical & Experimental Metastasis·Catherine Alix-Panabières, Klaus Pantel
Jul 18, 2020·Current Opinion in Oncology·Lisanne F van DesselMartijn P Lolkema
May 12, 2018·Diagnostics·Francesc Castro-GinerNicola Aceto
Aug 24, 2019·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Emma PaillerFrançoise Farace

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Methods Mentioned

BETA
PCR
Assay
Electrophoresis
chips
Feature Extraction

Software Mentioned

PyLOH
BEDTOOLS IntersectBed
FREEC
Control
BEDTOOLS suite
- FREEC ( Control - Free Copy number caller )
Ampli
Genomic Workbench
ABSOLUTE
R package

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