Construction of Whole Genomes from Scaffolds Using Single Cell Strand-Seq Data.

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.

Characterizing polymorphic inversions in human genomes by single-cell sequencing.

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.

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid genomes along the entire length of all chromosomes. We demonstrate this by building a complete haplotype for a HapMap individual (NA12878) at high accuracy (concordance 99.3%), without using generational information or statistical inference. By use of this approach, we mapped all meiotic recombination events in a family trio with high resolution (median range ∼14 kb) and phased larger structural variants like deletions, indels, and balanced rearrangements like inversions. Lastly, the single-cell resolution of Strand-seq allowed us to observe loss of heterozygosity regions in a small number of cells, a significant advantage for studies of heterogeneous cell populations, such as cancer cells. We conclude that Strand-seq is a unique and powerful approach to completely phase individual genomes and map inheritance patterns in families, while preserving haplotype differences between single cells.

Identification of sister chromatids by DNA template strand sequences.

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

Strand-seq: a unifying tool for studies of chromosome segregation.

Non random segregation of sister chromatids has been implicated to help specify daughter cell fate (the Silent Sister Hypothesis [1]) or to protect the genome of long-lived stem cells (the Immortal Strand Hypothesis [2]). The idea that sister chromatids are non-randomly segregated into specific daughter cells is only marginally supported by data in sporadic and often contradictory studies. As a result, the field has moved forward rather slowly. The advent of being able to directly label and differentiate sister chromatids in vivo using fluorescence in situ hybridization [3] was a significant advance for such studies. However, this approach is limited by the need for large tracks of unidirectional repeats on chromosomes and the reliance on quantitative imaging of fluorescent probes and rigorous statistical analysis to discern between the two competing hypotheses. A novel method called Strand-seq which uses next-generation sequencing to assay sister chromatid inheritance patterns independently for each chromosome [4] offers a comprehensive approach to test for non-random segregation. In addition Strand-seq enables studies on the deposition of chromatin marks in relation to DNA replication. This method is expected to help unify the field by testing previous claims of non-random segregation in an unbiased way in many model systems in vitro and in vivo.

DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution.

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm 9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint.

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph(p) mutants reveals an acceleration of S phase, showing that ph(p) regulates S phase length. Like ph(p) mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph(p) are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.

Stabilized coronavirus spike stem elicits a broadly protective antibody.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive ß-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.

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

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.

Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer's disease neurons.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease of the brain and the most common form of dementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has been proposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridization have shown that the brains of AD patients contain an increased number of aneuploid cells. However, because the reported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible role of aneuploidy in AD pathology. RESULTS: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assess aneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD (n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21 in all analyzed neuronal nuclei of a Down's syndrome sample (n = 36). Very low levels of aneuploidy were found in the brains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selective gain of chromosomes 17 or 21 in neurons of AD patients. CONCLUSION: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our results do not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to be confirmed by future studies in larger cohorts.

Looking beyond the post-genomic era.

A report on BioMed Central's fourth annual Beyond the Genome conference held at the University of California, San Francisco Mission Bay Conference Center, USA, 1-3 October 2013.

BAIT: Organizing genomes and mapping rearrangements in single cells.

Strand-seq is a single-cell sequencing technique to finely map sister chromatid exchanges (SCEs) and other rearrangements. To analyze these data, we introduce BAIT, software which assigns templates and identifies and localizes SCEs. We demonstrate BAIT can refine completed reference assemblies, identifying approximately 21 Mb of incorrectly oriented fragments and placing over half (2.6 Mb) of the orphan fragments in mm10/GRCm38. BAIT also stratifies scaffold-stage assemblies, potentially accelerating the assembling and finishing of reference genomes. BAIT is available at http://sourceforge.net/projects/bait/.

Epigenetic differences between sister chromatids?

Semi-conservative replication ensures that the DNA sequence of sister chromatids is identical except for replication errors and variation in the length of telomere repeats resulting from replicative losses and variable end processing. What happens with the various epigenetic marks during DNA replication is less clear. Many chromatin marks are likely to be copied onto both sister chromatids in conjunction with DNA replication, whereas others could be distributed randomly between sister chromatids. Epigenetic differences between sister chromatids could also emerge in a more predictable manner, for example, following processes that are associated with lagging strand DNA replication. The resulting epigenetic differences between sister chromatids could result in different gene expression patterns in daughter cells. This possibility has been difficult to test because techniques to distinguish between parental sister chromatids require analysis of single cells and are not obvious. Here, we briefly review the topic of sister chromatid epigenetics and discuss how the identification of sister chromatids in cells could change the way we think about asymmetric cell divisions and stochastic variation in gene expression between cells in general and paired daughter cells in particular.

Chromosome orientation fluorescence in situ hybridization to study sister chromatid segregation in vivo.

Previously, assays for sister chromatid segregation patterns relied on incorporation of 5-bromo-2'-deoxyuridine (BrdU) and indirect methods to infer segregation patterns after two cell divisions. In this study, we describe a method to differentially label sister chromatids of mouse cells and to directly assay sister chromatid segregation patterns after one cell division in vitro and in vivo by adaptation of the well-established CO-FISH technique. BrdU is incorporated into newly formed DNA strands, which are then subjected to photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially labeled sister chromatids in postmitotic cells are visualized using fluorescence microscopy, and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires 4 d for in vivo mouse tissues and 2 d for in vitro-cultured cells.