Highly multiplexed targeted DNA sequencing from single nuclei

Abstract

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48–96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5–6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer

“…Our study provides a comprehensive framework for studying the complexities of metastatic lineages that can be extended to many human cancer types. Such studies will soon become feasible as the cost and time for analyzing the genomes of thousands of single cells in parallel is realized through the development of new high-throughput technologies (Baslan et al 2015;Leung et al 2016;Zahn et al 2017;Vitak et al 2017). In the near future, the translation of these technologies into clinical practice will undoubtedly have a profound impact on reducing morbidity in cancer patients with metastatic disease.…”

“…1C). Single-cell libraries were barcoded and pooled together (48 cells) for copy number profiling using single-nucleus sequencing (SNS) (Navin et al 2011) or barcoded (96 cells) for highly multiplexed targeted sequencing using a 1000 cancer gene panel (T1000) that captures 12,500 exons and promoter regions (Leung et al 2016). The exome capture platform and the T1000 cancer gene panel only overlap within the exonic regions.…”

“…To resolve the clonal substructure of the primary and metastatic tumors, we applied highly multiplexed single-cell DNA sequencing (Leung et al 2016) to profile point mutations in 372 single cells using a 1000 cancer gene (T1000) panel. The single-cell sequencing data resulted in a mean coverage depth of 137× and average coverage breadth of 0.92 (Supplemental Table S4).…”

“…Our group reported the development of the first single-cell DNA sequencing method (single-nucleus sequencing) and used this method to delineate aneuploidy evolution in breast tumors (Navin et al 2011). Subsequent work from our group and others has led to the development of high-coverage single-cell sequencing methods to detect genome-wide mutations at base-pair resolution (Xu et al 2012;Zong et al 2012;Wang et al 2014;Leung et al 2015Leung et al , 2016Wang and Navin 2015;Gawad et al 2016). Computational methods can be used to infer phylogenetic trees from single-cell sequencing data (Davis and Navin 2016;Jahn et al 2016;Ross and Markowetz 2016).…”

A generic, cost-effective, and scalable cell lineage analysis platform

“…In addition, it would require much larger multiplex groups and/or utilization of additional AA chips, which also increases the cost per cell. Our future development is focused mainly on development and improving targeted enrichment one-pot multiplexed reactions, such as molecular inversion probes (MIP) or targeted capture (Leung et al 2016), in which per-probe cost is significantly lower due to multiplexed synthesis in advanced microarray technologies. MIPs were previously validated for low-scale MS analysis (Carlson et al 2015) and for a large number of targets (Li et al 2009), proving the feasibility of such protocols for massive MS analysis from SC WGA products.…”