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

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

Disseminated tumour cells with highly aberrant genomes are linked to poor prognosis in operable oesophageal adenocarcinoma

Background: Chromosomal instability (CIN) has repeatedly been identified as a prognostic marker. Here we evaluated the percentage of aberrant genome per cell (PAG) as a measure of CIN in single disseminated tumour cells (DTC) isolated from patients with operable oesophageal adenocarcinoma (EAC), to assess the impact of CINhigh DTCs on prognosis. Methods: We isolated CK18(positive) DTCs from bone marrow (BM) or lymph node (LN) preparations of operable EAC patients. After whole-genome amplification, single DTCs were analysed for chromosomal gains and losses using metaphase-based comparative genomic hybridisation (mCGH). We calculated the PAG for each DTC and determined the critical threshold value that identifies high-risk patients by STEPP (Subpopulation Treatment Effect Pattern Plot) analysis in two independent EAC patient cohorts (cohort #1, n = 44; cohort # 2; n = 29). Results: The most common chromosomal alterations observed among the DTCs were typical for EAC, but the DTCs showed a varying PAG between individual patients. Generally, LNDTCs displayed a significantly higher PAG than BMDTCs. STEPP analysis revealed an increasing PAG of DTCs to be correlated with an increased risk for short survival in two independent EAC cohorts as well as in the corresponding pooled analysis. In all three data sets (cohort # 1, cohort # 2 and pooled cohort), PAG(high) DTCs conferred an independent risk for a significantly decreased survival. Conclusions: The analysis of PAG/CIN in solitary marker-positive DTCs identifies operable EAC patients with poor prognosis, indicating a more aggressive minimal residual disease