Optimizing sparse sequencing of single cells for highly multiplex copy number profiling

Abstract

Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.

Biophysical technologies for understanding circulating tumor cell biology and metastasis

“…This is now possible on a single cell basis at the level of whole genome copy number analysis (63)(64)(65)(66)(67). Combined with morphologic analysis of CTCs, the addition of genomic analysis provides a capacity to study cancer populations as a collection of individual cells, often revealing cancers to be oligoclonal proliferations of cancer cells, rather than truly clonal populations possessing a singular genome (68).…”

The first five years of single-cell cancer genomics and beyond

“…To address these issues, several studies have begun to develop multiplexing strategies for SCS experiments. A recent method, called C-DOP-P, uses 96 barcodes to improve multiplexing of SNS and increase the throughput for single-cell copy number profiling (Baslan et al 2015). Commercial microfluidic approaches such as the Fluidigm C1 system have also been developed for analyzing 96 single-cell RNA profiles in parallel using nanoliter reaction volumes.…”

Construction of thousands of single cell genome sequencing libraries using combinatorial indexing

“…Single cell genome sequencing studies have taken one of two approaches: high depth of sequencing per cell for purposes of single nucleotide variant detection 2,8 , or low-pass sequencing to identify copy number variants (CNVs) and the presence of aneuploidy 1,9,10 . In the latter approach, the lack of a method to cost-effectively produce high numbers of single cell libraries has prevented the ability to quantitatively measure the frequency of CNV-harboring cells at population-level scale, or provide a robust analysis of heterogeneity in the context of cancer 11 .…”