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

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.

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Figure 1: Overview of Strand-seq protocol.
Figure 2: Generation of directional template strand sequencing libraries.
Figure 3: Cell sorting gates based on Hoechst quenching.
Figure 4: Size selection of final Strand-seq library pool.
Figure 5: Important parameters when considering Strand-seq libraries.
Figure 6: Overview of genomic features evident from a Strand-seq experiment.
Figure 7: Probabilities for template strand changes to occur in the same vicinity between libraries.

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Acknowledgements

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.

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Authors

Contributions

E.F. and A.D.S. developed and optimized the protocol. M.H. and A.D.S. designed bioinformatic analysis tools. D.C.J.S. established the protocol in a sister laboratory. A.D.S., M.H., D.C.J.S. and P.M.L. wrote the manuscript.

Corresponding author

Correspondence to Peter M Lansdorp.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Adapter and PCR primer oligonucleotides.

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’.

Supplementary Figure 2 PCR amplification reaction.

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.

Supplementary Figure 3 Size-selection using AMPure XP beads.

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.

Supplementary Figure 4 Set up of magnetic bead separation block.

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).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Methods. (PDF 1615 kb)

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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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