Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ~3 d from flow cytometry to sequence-ready DNA libraries.
At a glance
Sequence Read Archive
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- Supplementary Figure 1 (1 MB)
Outline of the informatics section. Provide a concise outline of all the steps with input-program-output labels.
- Supplementary Data (78 MB)
Input/output data for the informatics procedure of the protocol required to reproduce copy number profiles from single cell sequencing data. Supplementary Data Legend outlines all the data with descriptions.
- Supplementary Methods (107 KB)
Contains all programs required to process single cell sequencing data for copy number determination, from genome preparation to sequence mapping and copy number estimation. Supplementary Methods Legend outlines all the programs and their utilities.
- Supplementary Note (2 KB)
Descriptions of data and program files. Text providing a brief overview of the data and program files included in the Supplementary Methods and Supplementary Data.