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.

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.

Assembling draft genomes using contiBAIT.

SUMMARY: Massively parallel sequencing is now widely used, but data interpretation is only as good as the reference assembly to which it is aligned. While the number of reference assemblies has rapidly expanded, most of these remain at intermediate stages of completion, either as scaffold builds, or as chromosome builds (consisting of correctly ordered, but not necessarily correctly oriented scaffolds separated by gaps). Completion of de novo assemblies remains difficult, as regions that are repetitive or hard to sequence prevent the accumulation of larger scaffolds, and create errors such as misorientations and mislocalizations. Thus, complementary methods for determining the orientation and positioning of fragments are important for finishing assemblies. Strand-seq is a method for determining template strand inheritance in single cells, information that can be used to determine relative genomic distance and orientation between scaffolds, and find errors within them. We present contiBAIT, an R/Bioconductor package which uses Strand-seq data to repair and improve existing assemblies. AVAILABILITY AND IMPLEMENTATION: contiBAIT is available on Bioconductor. Source files available from GitHub. CONTACT: koneill@bcgsc.ca or mark.hills@stemcell.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

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.

Telomere maintenance through recruitment of internal genomic regions.

Cells surviving crisis are often tumorigenic and their telomeres are commonly maintained through the reactivation of telomerase. However, surviving cells occasionally activate a recombination-based mechanism called alternative lengthening of telomeres (ALT). Here we establish stably maintained survivors in telomerase-deleted Caenorhabditis elegans that escape from sterility by activating ALT. ALT survivors trans-duplicate an internal genomic region, which is already cis-duplicated to chromosome ends, across the telomeres of all chromosomes. These 'Template for ALT' (TALT) regions consist of a block of genomic DNA flanked by telomere-like sequences, and are different between two genetic background. We establish a model that an ancestral duplication of a donor TALT region to a proximal telomere region forms a genomic reservoir ready to be incorporated into telomeres on ALT activation.

Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes.

Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres.

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/.

Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells.

Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres and ALT-associated characteristics were induced after incorporation of the C-type variant repeat by a mutant telomerase. We propose that the presence of variant repeats throughout ALT telomeres results from recombination-mediated telomere replication and spreading of variant repeats from the proximal regions of the telomeres and that the consequent binding of nuclear receptors alters the architecture of telomeres to facilitate further recombination.

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.

Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae.

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.

Telomere length measurement-caveats and a critical assessment of the available technologies and tools.

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.

Short telomeres resulting from heritable mutations in the telomerase reverse transcriptase gene predispose for a variety of malignancies.

Telomeres are composed of long arrays of TTAGGG repeats and associated proteins that act as a protective cap for chromosome ends. The length of telomere repeats is set in the germline but decreases in somatic cells, primarily as a function of DNA replication. Progressive telomere shortening limits stem cell divisions and probably acts as a tumor suppressor mechanism. Using a sensitive PCR method to detect the length of individual telomere repeats on specific chromosomes, we confirmed that telomere length decreases from primitive to more differentiated human cell types within the hematopoietic hierarchy. Genetic mutations in the components of telomerase (the RNA template sequence hTERC, reverse transcriptase hTERT, and Syskerin DKC1) have recently been implicated in a variety of bone marrow failure syndromes, idiopathic pulmonary fibrosis, and more recently, acute myeloid leukemia (AML). The majority of mutations discovered in AML patients were heritable and resulted in partial loss of telomerase activity, a finding counterintuitive to the requirement of telomerase in cancer cells. We have found heritable hypomorphic TERT mutations in other cancers as well, and we propose that such mutations result in short telomeres and premature loss of stem cells. Loss of normal stem cells could provide strong selection for abnormal cells incapable of responding to DNA damage signals originating from short telomeres. Such cells will have a DNA repair defect resulting in genomic instability and a mutator phenotype. Our findings point to an intimate connection between senescence and cancer and highlight the important role of telomeres in the biology of normal and malignant human cells.

Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline.

A number of different processes that impact on telomere length dynamics have been identified but factors that affect the turnover of repeats located proximally within the telomeric DNA are poorly defined. We have identified a particular repeat type (CTAGGG) that is associated with an extraordinarily high mutation rate (20% per gamete) in the male germline. The mutation rate is affected by the length and sequence homogeneity of the (CTAGGG)n array. This level of instability was not seen with other sequence-variant repeats, including the TCAGGG repeat type that has the same composition. Telomeres carrying a (CTAGGG)n array are also highly unstable in somatic cells with the mutation process resulting in small gains or losses of repeats that also occasionally result in the deletion of the whole (CTAGGG)n array. These sequences are prone to quadruplex formation in vitro but adopt a different topology from (TTAGGG)n (see accompanying article). Interestingly, short (CTAGGG)2 oligonucleotides induce a DNA damage response (gammaH2AX foci) as efficiently as (TTAGGG)2 oligos in normal fibroblast cells, suggesting they recruit POT1 from the telomere. Moreover, in vitro assays show that (CTAGGG)n repeats bind POT1 more efficiently than (TTAGGG)n or (TCAGGG)n. We estimate that 7% of human telomeres contain (CTAGGG)n repeats and when present, they create additional problems that probably arise during telomere replication.

Probing the mitotic history and developmental stage of hematopoietic cells using single telomere length analysis (STELA).

In most human somatic cells, telomeres shorten as a function of DNA replication. Telomere length is therefore an indirect measure of the replicative history of cells. We measured the telomere lengths at XpYp chromosomes in purified human hematopoietic populations enriched for stem cells (Lin(-)CD34(+)CD38(-)Rho(-)) and successively more mature cells. The average telomere length showed expected length changes, pointing to the utility of this method for classifying novel differentiation markers. Interestingly, the frequency of abruptly shortened telomeres increased in terminally differentiated adult populations, suggesting that damage to telomeric DNA occurs or is not repaired upon hematopoietic differentiation. When Lin(-)CD34(+)CD38(-)Rho(-) cord blood cells were transplanted into immunodeficient mice, the telomeres of the most primitive regenerated human hematopoietic cells lost approximately 3 kb, indicative of more than 30 cell divisions. Further losses in differentiating cells were similar to those observed in pretransplantation cell populations. These results indicate extensive self-renewal divisions of human hematopoietic stem cells are the primary cause of telomere erosion upon transplantation rather than added cell divisions in downstream progenitors.

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia.

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Telomere elongation involves intra-molecular DNA replication in cells utilizing alternative lengthening of telomeres.

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation.

Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34(+) subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite.

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.

Multiple roles of the candidate oncogene ZNF217 in ovarian epithelial neoplastic progression.

The transcription factor ZNF217 is often amplified in ovarian cancer, but its role in neoplastic progression is unknown. We introduced ZNF217-HA by adenoviral and retroviral infection into normal human ovarian surface epithelial cells (OSE), i.e., the source of ovarian cancer, and into SV40 Tag/tag expressing, p53/pRB-deficient OSE with extended but finite life spans (IOSE). In OSE, ZNF217-HA reduced cell-substratum adhesion and accelerated loss of senescent cells, but caused no obvious proneoplastic changes. In contrast, ZNF217-HA transduction into IOSE yielded two permanent lines, I-80RZ and I-144RZ, which exhibited telomerase activity, stable telomere lengths, anchorage independence and reduced serum dependence, but were not tumorigenic in SCID mice. This immortalization required short-term EGF treatment near the time of crisis. The permanent lines were EGF-independent, but ZNF217-dependent since siRNA to ZNF217 inhibited anchorage independence and arrested growth. Array CGH revealed genomic changes resembling those of ovarian carcinomas, such as amplicons at 3q and 20q, and deletions at 4q and 18, associated with underexpressed annexin A10, N-cadherin, desmocollin 3 and PAI-2, which have been reported as tumor suppressors. The lines overexpressed EEF1A2, SMARA3 and STAT1 and underexpressed other oncogenes, tumor suppressors and extracellular matrix/adhesion genes. The results implicate ZNF217 as an ovarian oncogene, which is detrimental to senescing normal OSE cells but contributes to neoplastic progression in OSE with inactivated p53/RB. The resemblance of the genomic changes in the ZNF217-overexpressing lines to ovarian carcinomas provides a unique model to investigate interrelationships between these changes and ovarian neoplastic phenotypes.