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.
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Mills, R.E. et al. Mapping copy number variation by population-scale genome sequencing. Nature 470, 59–65 (2011).
Sudmant, P.H. et al. An integrated map of structural variation in 2,504 human genomes. Nature 526, 75–81 (2015).
Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013).
Ciriello, G. et al. Emerging landscape of oncogenic signatures across human cancers. Nat. Genet. 45, 1127–1133 (2013).
Alkan, C., Coe, B.P. & Eichler, E.E. Genome structural variation discovery and genotyping. Nat. Rev. Genet. 12, 363–376 (2011).
Feuk, L. Inversion variants in the human genome: role in disease and genome architecture. Genome Med. 2, 11 (2010).
Sindi, S.S. & Raphael, B.J. Identification of structural variation. in Genome Analysis: Current Procedures and Applications (ed. Poptsova, M.S.), (Caister Academic Press, 2014).
Korbel, J.O. et al. PEMer: a computational framework with simulation-based error models for inferring genomic structural variants from massive paired-end sequencing data. Genome Biol. 10, R23 (2009).
Ye, K., Schulz, M.H., Long, Q., Apweiler, R. & Ning, Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25, 2865–2871 (2009).
Rausch, T. et al. DELLY: structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics 28, i333–i339 (2012).
Xie, C. & Tammi, M.T. CNV-seq, a new method to detect copy number variation using high-throughput sequencing. BMC Bioinformatics 10, 80 (2009).
Voet, T. et al. Single-cell paired-end genome sequencing reveals structural variation per cell cycle. Nucleic Acids Res. 41, 6119–6138 (2013).
Nagano, T. et al. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell. Nat. Protoc. 10, 1986–2003 (2015).
Leung, M.L. et al. Highly multiplexed targeted DNA sequencing from single nuclei. Nat. Protoc. 11, 214–235 (2016).
Bakker, B. et al. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies. Genome Biol. 17, 115 (2016).
van den Bos, H. et al. Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer's disease neurons. Genome Biol. 17, 116 (2016).
Navin, N.E. The first five years of single-cell cancer genomics and beyond. Genome Res. 25, 1499–1507 (2015).
Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011).
Macaulay, I.C. & Voet, T. Single cell genomics: advances and future perspectives. PLoS Genet. 10, e1004126 (2014).
Falconer, E. et al. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution. Nat. Methods 9, 1107–1112 (2012).
Sanders, A.D. et al. Characterizing polymorphic inversions in human genomes by single cell sequencing. Genome Res. 26, 1575–1587 (2016).
Hills, M., O'Neill, K., Falconer, E., Brinkman, R. & Lansdorp, P.M. BAIT: organizing genomes and mapping rearrangements in single cells. Genome Med. 5, 82 (2013).
Porubsky, D. et al. Building complete chromosomal haplotypes using single-cell sequencing. Genome Res. 26, 1565–1574 (2016).
Falconer, E., Chavez, E., Henderson, A. & Lansdorp, P.M. Chromosome orientation fluorescence in situ hybridization to study sister chromatid segregation in vivo. Nat. Protoc. 5, 1362–1377 (2010).
Smith, J.R. & Pereira-Smith, O.M. Replicative senescence: implications for in vivo aging and tumor suppression. Science 273, 63–67 (1996).
Rabinovitch, P.S. Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry. Proc. Natl. Acad. Sci. USA 80, 2951–2955 (1983).
Poot, M., Silber, J.R. & Rabinovitch, P.S. A novel flow cytometric technique for drug cytotoxicity gives results comparable to colony-forming assays. Cytometry 48, 1–5 (2002).
Latt, S.A., George, Y.S. & Gray, J.W. Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst. J. Histochem. Cytochem. 25, 927–934 (1977).
Kubbies, M. & Rabinovitch, P.S. Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes. Cytometry 3, 276–281 (1983).
Fogt, F. et al. Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine. Cytometry 12, 33–41 (1991).
Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Lansdorp, P.M., Falconer, E., Tao, J., Brind'Amour, J. & Naumann, U. Epigenetic differences between sister chromatids? Ann. N. Y. Acad. Sci. 1266, 1–6 (2012).
This work was supported by grants from the Canadian Institutes of Health Research (RMF-92093 and 105265), the US National Institutes of Health (R01GM094146) and the Terry Fox Foundation (018006), as well as an Advanced Grant (Nr 294740) from the European Research Council to P.M.L., and a Vanier Graduate Scholarship from the Natural Science and Engineering Research Council to A.D.S.
The authors declare no competing financial interests.
Integrated supplementary information
Sequence composition of the A) Illumina PE Adapters and B) PCR Primers required for the protocol. Following reconstitution, the adapters are annealed to generate a forked product. An internal hexamer barcode is included in the custom multiplexing primer PE 2.0, as indicated by ‘N’.
Inputs into the reaction mix are adapter ligation products (template strand as solid box, nicked BrdU strand as fragmented box) and PCR primers (Illumina PE 1.0 and custom multiplexing PE 2.0). The first round of amplification involves only primer PE 2.0, which anneals to the A1 adapter to initiate elongation (dashed blue line) and introduce the multiplexing barcode (red bar, labeled ‘B’) at the 3' end of the template molecule. The polymerase stalls on the fragmented BrdU-positive molecule (indicated with a blue square), terminating synthesis. The subsequent rounds of amplification (17 cycles in total) are exponential and involve both primers. The template strand is preferentially amplified during this reaction to produce a directional sequencing library. Apostrophes (e.g. A1’ or B’) are used to indicate complement DNA sequences.
60ng of a 50 bp DNA ladder was input and Agencourt AMPure XP bead clean-up was performed using bead ratios of either 1.8x or 0.8x. The bioanalyzer trace illustrates the size range of products enriched for at each bead: DNA ratio, with the number of basepair (bp) indicated above each peak. The expected lengths of products and byproducts generated during the Strand-seq protocol are listed.
A) A solid rubber gasket (arrow) is placed around the magnet to slightly lift the 384-well plate and ensure the bead pellet is positioned lower in the well. This allows lower elution volumes and subsequently lower enzyme reaction volumes. As illustrated in B) the bead pellet remains submerged when lifted by the gasket (picture right), which is not the case when no gasket is used (picture left).
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Sanders, A., Falconer, E., Hills, M. et al. Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs. Nat Protoc 12, 1151–1176 (2017). https://doi.org/10.1038/nprot.2017.029
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