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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 214-235 |
| Number of pages | 22 |
| Journal | Nature Protocols |
| Volume | 11 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 1 2016 |
Funding
acknoWleDGMents N.E.N. is a Nadia’s Gift Foundation Damon Runyon-Rachleff Innovator (DRR-25-13), and also is a T.C. Hsu Endowed Scholar. This work was supported by a gift from the Eric & Liz Lefkofsky Family Foundation. The study was supported by grants to N.E.N. from the National Cancer Institute (NCI; no. 1RO1CA169244-01), the National Institutes of Health (NIH; no. R21CA174397-01) and an Agilent University Relations Grant. This work was supported by the MD Anderson Cancer Moonshot Knowledge Gap Award, Center for Genetics & Genomics and Center for Epigenetics. M.L.L. is supported by a Research Training Award from the Cancer Prevention and Research Institute of Texas (CPRIT RP140106), and is also supported by the American Legion Auxiliary (ALA) and Hearst Foundations. This work was also supported by the MD Anderson Sequencing Core Facility Grant (no. CA016672) and the Flow Cytometry Facility grant from NIH (no. CA016672). C.K. is supported by the NIH National Center for Advancing Translational Sciences (TL1TR000369 and UL1TR000371) and the ALA. This work was supported by a CPRIT research training award to J.J. (RP101502). We thank F. Meric-Bernstam and K. Eterovic for their support with the cancer gene targeted capture panels. We also thank L. Ramagli, K. Khanna, E. Thompson and H. Tang at the MD Anderson Sequencing Core Facility for supporting the sequencing experiments. We are also grateful to W. Schober and N. Patel at the MD Anderson Flow Core Facility for their support.
ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology
