Characterizing polymorphic inversions in human genomes by single-cell sequencing

Abstract

Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive phenotypes and disease susceptibilities. For this, single-cell studies are paramount, as it becomes increasingly clear that the contribution of rare but functional cellular subpopulations is important for disease prognosis, management, and progression. Until now, studying these associations has been challenged by our inability to map structural rearrangements accurately and comprehensively. To overcome this, we coupled single-cell sequencing of DNA template strands (Strand-seq) with custom analysis software to rapidly discover, map, and genotype genomic rearrangements at high resolution. This allowed us to explore the distribution and frequency of inversions in a heterogeneous cell population, identify several polymorphic domains in complex regions of the genome, and locate rare alleles in the reference assembly. We then mapped the entire genomic complement of inversions within two unrelated individuals to characterize their distinct inversion profiles and built a nonredundant global reference of structural rearrangements in the human genome. The work described here provides a powerful new framework to study structural variation and genomic heterogeneity in single-cell samples, whether from individuals for population studies or tissue types for biomarker discovery.

Direct chromosome-length haplotyping by single-cell sequencing

“…By sequencing only the original template strand of the inherited chromatids, we can distinguish both homologs in a single cell as two Crick template strands (CC), two Watson templates (WW), or a combination of Watson and Crick templates (WC) (Fig. 1A, iii;Falconer et al 2012;Hills et al 2013;Sanders et al 2016).…”

“…2, black asterisks). These switches most likely represent homozygous inversions in these regions (Sanders et al 2016).…”

“…Such a short switch in haplotypes could result from homozygous inversions, from two independent meiotic crossovers in close vicinity, or from a gene conversion event. We examined the template strand directionality of this region and did not find evidence supporting an inversion (Sanders et al 2016), suggesting this represents either a meiotic or a conversion event. We located 18 additional regions in the child's homologs that exhibited a short haplotype switch involving at least three consecutive heterozygous SNV positions (Supplemental Table S6).…”

“…S9B, arrowheads). Importantly, balanced rearrangements like inversions that are difficult to detect using current technologies can be reliably mapped and phased using Strand-seq (Sanders et al 2016). To our knowledge, Strand-seq is the only technique able to simultaneously map and phase heterozygous inversions (Supplemental Fig.…”

“…For this, we scanned the genome of each single cell and counted the number of Crick (forward; "+") and Watson (reverse; "−") reads in equally sized regions (default, 1 Mb). Fisher exact tests were used to calculate the probability that a region contained approximately equal numbers of Crick and Watson reads and agreed with the expected 50:50 ratio of a WC region (Sanders et al 2016). Subsequently only WC regions >5 Mb were selected for further analysis.…”