Single-cell template strand sequencing by strand-seq enables the characterization of individual homologs

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Item Type:	Article
Title:	Single-cell template strand sequencing by strand-seq enables the characterization of individual homologs
Creators Name:	Sanders, A.D., Falconer, E., Hills, M., Spierings, D.C.J. and Lansdorp, P.M.
Abstract:	The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5′–3′ orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU(+) strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.
Keywords:	Alleles, DNA Sequence Analysis, Single-Cell Analysis, Single-Stranded DNA, Staining and Labeling
Source:	Nature Protocols
ISSN:	1754-2189
Publisher:	Nature Publishing Group
Volume:	12
Number:	6
Page Range:	1151-1176
Date:	June 2017
Official Publication:	https://doi.org/10.1038/nprot.2017.029
PubMed:	View item in PubMed

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